Antibodies against n-acetylgucosamine and n-acetyl-galactosamine

ABSTRACT

It provides antibodies and chimeric antigen receptors (CARs) that specifically bind to an epitope containing N-acetyl-glucosamine and/or N-acetyl-galactosamine, e.g., expressed by a cancer cell or an inflammatory cell. Also provided are compositions including these antibodies and/or CARs, as well as polynucleotides, vectors, host cells, methods, and kits related thereto. Further provided are methods and kits for treating or preventing cancer in an individual by administering to the individual an antibody that specifically binds to an epitope containing N-acetylglucosamine or N-acetyl-galactosamine, or a T cell comprising a CAR that specifically binds to an epitope containing N-acetylglucosamine or N-acetyl-galactosamine, optionally in combination with another anti-cancer agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of International PatentApplication Serial No. PCT/CN2016/074146, filed Feb. 19, 2016, which ishereby incorporated by reference in its entirety.

FIELD

This invention relates to antibodies that specifically bindN-acetylglucosamine and/or N-acetyl-galactosamine, as well ascompositions, polynucleotides, vectors, host cells, methods ofproduction, methods of use, and kits related thereto.

BACKGROUND

Cancer is one of the most challenging disorders to treat in modernmedicine for several reasons. Since cancer arises from the abnormalbehavior of one's own cells, distinguishing cancer cells from normalcells within a patient is a difficult problem. Often the body's ownimmune system has difficulty identifying and eliminating cancerouscells. In addition, “cancer” refers to a constellation of individualdisorders, i.e., types and subtypes of cancer. Many different cell typescan become cancerous through many different mechanisms, resulting in atremendous phenotypic variety in the types of cancer cells that mayarise. This diversity is highly problematic for cancer treatment becausedifferent types of cancer cells may have different identifyingproperties for diagnosis, or they may possess different therapeuticweaknesses or resistant properties. This problem makes it difficult tocome up with ways to diagnose, treat, and/or prevent multiple types ofcancer through a single therapeutic strategy or agent. Even thoughoncology has advanced tremendously in the last decade, there is still aneed to identify new biomarkers specific to cancer cells, particularlybiomarkers that characterize multiple types of cancer but not normaltissues.

Cell surface molecules are highly important for cancer cells. Thesemolecules are critically involved in cell-cell interactions, which areimportant for many cancer cell behaviors, including cell invasion,metastasis, evasion of the immune system, and responses to therapeuticagents. Cancer cells are known to express many cell surface proteinsdifferently from normal cells. However, many cell surface proteins aremodified by the addition of saccharides (e.g., N-acetylglucosamine orN-acetyl-galactosamine), a process termed protein glycosylation. Howspecific cell surface proteins are modified by the addition ofsaccharides, which saccharides may be found on which cell surfaceproteins, and how patterns of glycosylation change during differenttypes or phases of carcinogenesis are all problems that are justbeginning to be explored (for a review, see Moremen, K. W., et al.(2012) Nat. Rev. Mol. Cell Biol. 13(7):448-62). This diversity inglycosylation increases the complexity of cancer cell recognition bysurface biomarkers.

Therefore, there remains a need for new biomarkers and therapeuticagents useful in the diagnosis, treatment, and prevention of cancer,particularly for biomarkers and agents that target cancer-specificpatterns of glycosylation. In particular, there remains a need forimproved diagnostic and/or therapeutic agents that specifically bind toone or more saccharides (e.g., N-acetylglucosamine and/orN-acetyl-galactosamine), which may be useful, inter alia, for thetreatment of cancer, inflammatory, and/or autoimmune diseases.

All references cited herein, including patent applications, patentpublications, and UniProtKB/Swiss-Prot Accession numbers are hereinincorporated by reference in their entirety, as if each individualreference were specifically and individually indicated to beincorporated by reference

BRIEF SUMMARY

To meet the demand for new biomarkers and therapeutic agents useful inthe diagnosis, treatment, and prevention of multiple types of cancer,disclosed herein are monoclonal antibodies that specifically bindN-acetylglucosamine or N-acetyl-galactosamine (e.g., expressed by acancer cell or an inflammatory cell), as well as compositions,polynucleotides, vectors, host cells, methods of production, and kitsrelated thereto. Further disclosed are methods of treating or preventingcancer in an individual by administering antibodies that specificallybind N-acetylglucosamine or N-acetyl-galactosamine expressed on a cellsurface of a cancer cell, and methods of diagnosing cancer usingantibodies that specifically bind N-acetylglucosamine orN-acetyl-galactosamine. These compositions and methods are based in parton the surprising discovery that human cancer cells representing manydifferent types of cancer express higher levels of N-acetylglucosamineand/or N-acetyl-galactosamine than normal human tissues. Moreover, thepresent disclosure demonstrates the surprising result that antibodiesthat specifically bind N-acetylglucosamine and/or N-acetyl-galactosamineexpressed on a cell surface of a cancer cell are highly potent andeffective in reducing the growth rate of several diverse types of cancercells, both in vitro and in vivo. Additionally, the present applicationdescribes the unexpected finding that antibodies that specifically bindN-acetylglucosamine or N-acetyl-galactosamine may be used as aneffective preventative and treatment for gastrointestinal diseases, suchas viral infection, inflammatory bowel disease, and hemorrhoids, andrheumatoid arthritis.

In one aspect, provided herein are antibodies that specifically bind toan epitope comprising N-acetylglucosamine and/or N-acetyl-galactosamine.Also provided are compositions comprising these antibodies, as well aspolynucleotides, vectors, host cells, and methods useful in theproduction thereof. Further provided are methods and kits useful fortreating or preventing cancer in an individual by administering to theindividual an antibody that specifically binds to an epitope comprisingN-acetylglucosamine or N-acetyl-galactosamine, optionally in combinationwith another anti-cancer agent. Further provided are methods and kitsuseful for treating or preventing gastrointestinal disease in anindividual by administering to the individual an antibody thatspecifically binds to an epitope comprising N-acetylglucosamine orN-acetyl-galactosamine. Yet further provided are methods and kits usefulfor detecting the presence of cancer cells in an individual by obtaininga biological sample from an individual, contacting the biological samplewith an antibody that specifically binds to an epitope comprisingN-acetylglucosamine and/or N-acetyl-galactosamine, and detecting theamount of antibody binding to the biological sample, where antibodybinding indicates the presence of cancer cells in the individual.

In certain aspects, the present disclosure provides an isolatedmonoclonal antibody that specifically binds to an epitope comprisingN-acetylglucosamine or N-acetyl-galactosamine, wherein the antibodycomprises a light chain variable region comprising an HVR-L1 sequence ofSEQ ID NO: 5, an HVR-L2 sequence of FTSX₁LX₂S (SEQ ID NO: 25), and anHVR-L3 sequence of SEQ ID NO: 9, wherein X₁ is T or S and X₂ is Q or E.In certain embodiments, the HVR-L2 sequence comprises the amino acidsequence of SEQ ID NO: 7. In certain embodiments, the antibody comprisesa light chain variable region comprising the amino acid sequence of SEQID NO: 13. In certain embodiments, the antibody comprises a light chaincomprising the amino acid sequence of SEQ ID NO: 16. In certainembodiments, the HVR-L2 sequence comprises the amino acid sequence ofSEQ ID NO: 8. In certain embodiments, the antibody comprises a lightchain variable region comprising the amino acid sequence of SEQ ID NO:14. In certain embodiments, the antibody comprises a light chaincomprising the amino acid sequence of SEQ ID NO: 17. In certainembodiments, the antibody further comprises a heavy chain variableregion comprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequenceof SEQ ID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3. In certainembodiments, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 10. In certainembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 11. In some embodiments, the antibodycomprises a heavy chain variable region comprising three HVRs of SEQ IDNO: 10 and/or a light chain variable region comprising three HVRs of SEQID NO: 12. In some embodiments, the antibody comprises a heavy chainvariable region comprising three HVRs of SEQ ID NO: 10 and/or a lightchain variable region comprising three HVRs of SEQ ID NO: 13. In someembodiments, the antibody comprises a heavy chain variable regioncomprising three HVRs of SEQ ID NO: 10 and/or a light chain variableregion comprising three HVRs of SEQ ID NO: 14. In some embodiments, theantibody comprises a heavy chain variable region comprising three HVRsof SEQ ID NO: 10 and/or a light chain variable region comprising threeHVRs of SEQ ID NO: 19. In some embodiments, the antibody comprises aheavy chain variable region comprising three HVRs of SEQ ID NO: 10and/or a light chain variable region comprising three HVRs of SEQ ID NO:23. In some embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 10 and/or alight chain variable region comprising the amino acid sequence of SEQ IDNO: 12. In some embodiments, the antibody comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 10and/or a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 13. In some embodiments, the antibody comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO:10 and/or a light chain variable region comprising the amino acidsequence of SEQ ID NO: 14. In some embodiments, the antibody comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 10 and/or a light chain variable region comprising the amino acidsequence of SEQ ID NO: 19. In some embodiments, the antibody comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 10 and/or a light chain variable region comprising the amino acidsequence of SEQ ID NO: 23. In certain embodiments that may be combinedwith any of the preceding embodiments, the antibody is an antibodyfragment. In certain embodiments, the antibody is a Fab fragment, scFv,minibody, diabody, scFv multimer, or bispecific antibody fragment. Incertain embodiments, the antibody is a humanized antibody. In certainembodiments, the antibody is a human antibody. In certain embodiments,the antibody is a chimeric antibody. In certain embodiments, theantibody specifically binds to an epitope comprising N-acetylglucosamineand an epitope comprising N-acetyl-galactosamine. In certain embodimentsthat may be combined with any of the preceding embodiments, the epitopeis expressed by a cancer cell or an inflammatory cell the epitope isexpressed on a cell surface of the cancer cell. In certain embodiments,the epitope is expressed in the cancer cell. In certain embodiments thatmay be combined with any of the preceding embodiments, the cancer cellis selected from a pancreatic adenocarcinoma cell, a colonadenocarcinoma cell, a rectal adenocarcinoma cell, an esophagealadenocarcinoma cell, a leukemia cell, an adenoid carcinoma cell, afibrosarcoma cell, a duodenal adenocarcinoma cell, a glioma cell, ahepatocarcinoma cell, a lung cancer cell, a breast cancer cell, aglioblastoma cell, an ovarian carcinoma cell, and a cervicaladenocarcinoma cell. In certain embodiments, the pancreaticadenocarcinoma cell is a pancreatic ductal adenocarcinoma cell. Incertain embodiments, the binding of the antibody to the epitope inhibitsgrowth of the cancer cell. In certain embodiments, the lung cancer cellis a small cell lung cancer cell, a lung squamous cell carcinoma cell,or a lung adenocarcinoma cell. In certain embodiments, the inflammatorycell is an intestinal inflammatory cell of colitis, inflammatory boweldisease, or gastroenteritis, and the epitope is expressed on a cellsurface of the inflammatory cell. In certain embodiments, theinflammatory cell is an inflammatory cell of rheumatoid arthritis, andthe epitope is expressed on a cell surface of the inflammatory cell. Incertain embodiments, the rheumatoid arthritis is induced by collagen.

In further aspects, the present disclosure provides an isolatedpolynucleotide comprising a nucleic acid sequence encoding an antibodyaccording to any of the above embodiments. In still further aspects, thepresent disclosure provides a vector comprising a nucleic acid sequenceencoding an antibody according to any of the above embodiments. In yetstill further aspects, the present disclosure provides an isolated hostcell comprising an isolated polynucleotide comprising a nucleic acidsequence encoding an antibody according to any of the above embodimentsor a vector comprising a nucleic acid sequence encoding an antibodyaccording to any of the above embodiments. In yet still further aspects,the present disclosure provides an isolated host cell comprising anisolated polynucleotide comprising a nucleic acid sequence encoding anantibody according to any of the above embodiments or a vectorcomprising a nucleic acid sequence encoding an antibody according to anyof the above embodiments. In yet still further aspects, the presentdisclosure provides methods of producing an antibody, comprisingculturing a host cell according to any of the above embodiments, thatproduces the antibody according to any of the above embodiments, andrecovering the antibody from the cell culture. In yet still furtheraspects, the present disclosure provides an antibody produced by themethods of producing an antibody according to any of the aboveembodiments. In other aspects, the present disclosure provides acomposition comprising an antibody according to any of the aboveembodiments, and a pharmaceutically acceptable carrier.

In other aspects, the present disclosure provides a method for treatingor preventing cancer in an individual, comprising administering to theindividual an effective amount of a composition comprising an antibodyaccording to any of the above embodiments. In other aspects, the presentdisclosure provides a method for treating or preventing cancer in anindividual, comprising administering to the individual an amount of anantibody according to any of the above embodiments, and an amount ofanother anti-cancer agent, where the antibody and the anti-cancer agentin conjunction provide effective treatment or prevention of cancer inthe individual. In some embodiments, the individual has, or has beendiagnosed with, cancer. In certain embodiments, the cancer is selectedfrom pancreatic cancer, colon cancer, rectal cancer, esophageal cancer,leukemia, adenocarcinoma, fibrosarcoma, duodenal adenocarcinoma, braincancer, liver cancer, lung cancer, breast cancer, ovarian cancer, andcervical cancer. In certain embodiments, the individual is a human. Incertain embodiments, the individual is a non-human animal. In certainembodiments, the anti-cancer agent is a chemotherapeutic agent.

In other aspects, the present disclosure provides a method for detectingcancer cells in an individual, comprising contacting a biological samplefrom the individual with an antibody according to any of the aboveembodiments and detecting binding of the antibody to the biologicalsample, where binding of the antibody to the sample may indicate thepresence of cancer cells in the individual. In certain embodiments, themethod further comprises comparing the amount of antibody bindingdetected with an amount of antibody binding to a control sample. Incertain embodiments that may be combined with any of the precedingembodiments, the binding of the antibody to the biological sample isdetected by an assay selected from an ELISA assay, a flow cytometryassay, an immunohistochemistry assay, an immunofluorescence assay, acirculating tumor cells assay, and an immune-colloidal gold assay. Incertain embodiments that may be combined with any of the precedingembodiments, the biological sample is selected from blood, serum, urine,feces, milk, semen, saliva, chest fluid, abdominal fluid, cerebrospinalfluid, sputum, and any other body fluid or secretion. In certainembodiments, the individual is a human. In certain embodiments, theindividual is a non-human animal. In certain embodiments that may becombined with any of the preceding embodiments, the cancer cells areselected from pancreatic cancer cells, leukemia cells, adenocarcinomacells, fibrosarcoma cells, lung cancer cells, liver cancer cells, breastcancer cells, colon or colorectal cancer cells, esophageal cancer cells,stomach cancer cells, endometrial cancer cells, cervical cancer cells,thyroid cancer cells, brain cancer cells, and lymphoma cells.

In other aspects, the present disclosure provides a method for treatingor preventing gastrointestinal disease in an individual comprisingadministering to the individual an effective amount of an antibodyaccording to any of the above embodiments. In some embodiments, theindividual has, or has been diagnosed with, a gastrointestinal diseaseof the present disclosure. In certain embodiments, the individual hasinflammatory bowel disease. In certain embodiments, the individual hasCrohn's disease. In certain embodiments, the individual has ulcerativecolitis. In certain embodiments, the individual has acute infectiousgastroenteritis. In certain embodiments, the individual has ahemorrhoid. In certain embodiments, the individual has agastrointestinal disease caused by a viral infection. In certainembodiments, the viral infection is a rotaviral infection or a porcineepidemic diarrhea viral infection. In certain embodiments that may becombined with any of the preceding embodiments, the individual is ahuman. In certain embodiments that may be combined with any of thepreceding embodiments, the individual is a non-human animal. In certainembodiments that may be combined with any of the preceding embodiments,the antibody is administered intravenously, intramuscularly,subcutaneously, topically, orally, transdermally, intraperitoneally,intraorbitally, by implantation, by inhalation, intrathecally,intraventricularly, or intranasally.

In other aspects, the present disclosure provides a method for treatingor preventing rheumatoid arthritis in an individual comprisingadministering to the individual an effective amount of an antibody thatspecifically binds to an epitope comprising N-acetylglucosamine orN-acetyl-galactosamine. In some embodiments, the individual has, or hasbeen diagnosed with, rheumatoid arthritis. In some embodiments, theepitope is expressed by an inflammatory cell. In certain embodiments,the antibody specifically binds to an epitope comprisingN-acetylglucosamine and an epitope comprising N-acetyl-galactosamine. Incertain embodiments, the antibody is an antibody fragment. In certainembodiments, the antibody is a Fab fragment, scFv, minibody, diabody,scFv multimer, or bispecific antibody fragment. In certain embodiments,the antibody is a humanized antibody. In certain embodiments, theantibody is a human antibody. In certain embodiments, the antibody is achimeric antibody. In certain embodiments, the antibody comprises aheavy chain variable region comprising three HVRs from the amino acidsequence of SEQ ID NO:10, and a light chain variable region comprisingthree HVRs from an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 12, 13, 14, 19, and 23. In some embodiments,the antibody comprises a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 10 and/or a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 12. In someembodiments, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 10 and/or a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 13. Insome embodiments, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 10 and/or a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 14. Insome embodiments, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 10 and/or a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 19. Insome embodiments, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 10 and/or a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 23Incertain embodiments, the antibody comprises a heavy chain variableregion comprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequenceof SEQ ID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3; and a lightchain variable region comprising an HVR-L1 sequence of SEQ ID NO: 4, anHVR-L2 sequence of SEQ ID NO: 6, and an HVR-L3 sequence of SEQ ID NO: 9.In certain embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:10, and a lightchain variable region comprising the amino acid sequence of SEQ IDNO:12. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:11, and a light chaincomprising the amino acid sequence of SEQ ID NO:15. In certainembodiments, the antibody comprises a heavy chain variable regioncomprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3; and a light chainvariable region comprising an HVR-L1 sequence of SEQ ID NO: 21, anHVR-L2 sequence of SEQ ID NO: 22, and an HVR-L3 sequence of SEQ ID NO:9. In certain embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:10, and a lightchain variable region comprising the amino acid sequence of SEQ IDNO:23. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:11, and a light chaincomprising the amino acid sequence of SEQ ID NO:24. In certainembodiments, the antibody comprises a heavy chain variable regioncomprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3; and a light chainvariable region comprising an HVR-L1 sequence of SEQ ID NO: 4, an HVR-L2sequence of SEQ ID NO: 18, and an HVR-L3 sequence of SEQ ID NO: 9. Incertain embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:10, and a lightchain variable region comprising the amino acid sequence of SEQ IDNO:19. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:11, and a light chaincomprising the amino acid sequence of SEQ ID NO:20. In certainembodiments, the antibody comprises a heavy chain variable regioncomprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3; and a light chainvariable region comprising an HVR-L1 sequence of SEQ ID NO: 5, an HVR-L2sequence of SEQ ID NO: 7, and an HVR-L3 sequence of SEQ ID NO: 9. Incertain embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:10, and a lightchain variable region comprising the amino acid sequence of SEQ IDNO:13. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:11, and a light chaincomprising the amino acid sequence of SEQ ID NO:16. In certainembodiments, the antibody comprises a heavy chain variable regioncomprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3; and a light chainvariable region comprising an HVR-L1 sequence of SEQ ID NO: 5, an HVR-L2sequence of SEQ ID NO: 8, and an HVR-L3 sequence of SEQ ID NO: 9. Incertain embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:10, and a lightchain variable region comprising the amino acid sequence of SEQ IDNO:14. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:11, and a light chaincomprising the amino acid sequence of SEQ ID NO:17. In certainembodiments that may be combined with any of the preceding embodiments,the individual is a human. In certain embodiments that may be combinedwith any of the preceding embodiments, the individual is a non-humananimal. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody is administered intravenously,intramuscularly, subcutaneously, topically, orally, transdermally,intraperitoneally, intraorbitally, by implantation, by inhalation,intrathecally, intraventricularly, or intranasally.

In other aspects, the present disclosure provides a kit comprising apharmaceutical composition comprising an antibody according to any ofthe above embodiments. In certain aspects, the kit further comprisesinstructions for administering an effective amount of the pharmaceuticalcomposition to an individual for treating or preventing cancer. In someembodiments, the individual has, or has been diagnosed with, cancer. Incertain aspects, the kit further comprises instructions foradministering an effective amount of the pharmaceutical composition toan individual for treating or preventing gastrointestinal disease. Insome embodiments, the individual has, or has been diagnosed with,gastrointestinal disease. In certain aspects, the kit further comprisesinstructions for administering an effective amount of the pharmaceuticalcomposition to an individual for treating or preventing an autoimmunedisease. In certain aspects, the kit further comprises instructions fordetecting the presence of cancer cells in an individual. In certainaspects, the kit further comprises instructions for determining a levelof N-acetylglucosamine or N-acetyl-galactosamine in a biological samplefrom an individual with cancer.

In other aspects, the present disclosure provides a kit comprising apharmaceutical composition comprising an antibody that specificallybinds to an epitope comprising N-acetylglucosamine orN-acetyl-galactosamine, wherein the epitope is expressed by aninflammatory cell; and instructions for administering an effectiveamount of the composition to an individual for treating or preventingrheumatoid arthritis. In some embodiments, the individual has, or hasbeen diagnosed with, rheumatoid arthritis. In certain embodiments, theantibody specifically binds to an epitope comprising N-acetylglucosamineand an epitope comprising N-acetyl-galactosamine. In certainembodiments, the antibody is an antibody fragment. In certainembodiments, the antibody is a Fab fragment, scFv, minibody, diabody,scFv multimer, or bispecific antibody fragment. In certain embodiments,the antibody is a humanized antibody. In certain embodiments, theantibody is a human antibody. In certain embodiments, the antibody is achimeric antibody. In certain embodiments, the antibody comprises aheavy chain variable region comprising three HVRs from the amino acidsequence of SEQ ID NO:10, and a light chain variable region comprisingthree HVRs from an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 12, 13, 14, 19, and 23. In certainembodiments, the antibody comprises a heavy chain variable regioncomprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3; and a light chainvariable region comprising an HVR-L1 sequence of SEQ ID NO: 4, an HVR-L2sequence of SEQ ID NO: 6, and an HVR-L3 sequence of SEQ ID NO: 9. Incertain embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:10, and a lightchain variable region comprising the amino acid sequence of SEQ IDNO:12. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:11, and a light chaincomprising the amino acid sequence of SEQ ID NO:15. In certainembodiments, the antibody comprises a heavy chain variable regioncomprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3; and a light chainvariable region comprising an HVR-L1 sequence of SEQ ID NO: 21, anHVR-L2 sequence of SEQ ID NO: 22, and an HVR-L3 sequence of SEQ ID NO:9. In certain embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:10, and a lightchain variable region comprising the amino acid sequence of SEQ IDNO:23. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:11, and a light chaincomprising the amino acid sequence of SEQ ID NO:24. In certainembodiments, the antibody comprises a heavy chain variable regioncomprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3; and a light chainvariable region comprising an HVR-L1 sequence of SEQ ID NO: 4, an HVR-L2sequence of SEQ ID NO: 18, and an HVR-L3 sequence of SEQ ID NO: 9. Incertain embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:10, and a lightchain variable region comprising the amino acid sequence of SEQ IDNO:19. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:11, and a light chaincomprising the amino acid sequence of SEQ ID NO:20. In certainembodiments, the antibody comprises a heavy chain variable regioncomprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3; and a light chainvariable region comprising an HVR-L1 sequence of SEQ ID NO: 5, an HVR-L2sequence of SEQ ID NO: 7, and an HVR-L3 sequence of SEQ ID NO: 9. Incertain embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:10, and a lightchain variable region comprising the amino acid sequence of SEQ IDNO:13. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:11, and a light chaincomprising the amino acid sequence of SEQ ID NO:16. In certainembodiments, the antibody comprises a heavy chain variable regioncomprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3; and a light chainvariable region comprising an HVR-L1 sequence of SEQ ID NO: 5, an HVR-L2sequence of SEQ ID NO: 8, and an HVR-L3 sequence of SEQ ID NO: 9. Incertain embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:10, and a lightchain variable region comprising the amino acid sequence of SEQ IDNO:14. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:11, and a light chaincomprising the amino acid sequence of SEQ ID NO:17. In certainembodiments that may be combined with any of the preceding embodiments,the individual is a human. In certain embodiments that may be combinedwith any of the preceding embodiments, the individual is a non-humananimal. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody is administered intravenously,intramuscularly, subcutaneously, topically, orally, transdermally,intraperitoneally, intraorbitally, by implantation, by inhalation,intrathecally, intraventricularly, or intranasally.

In other aspects, the present disclosure provides a chimeric antigenreceptor (CAR) that comprises an antibody fragment comprising a lightchain variable region comprising an HVR-L1 sequence of SEQ ID NO: 5, anHVR-L2 sequence of SEQ ID NO: 7, and an HVR-L3 sequence of SEQ ID NO: 9and/or a heavy chain variable region comprising an HVR-H1 sequence ofSEQ ID NO: 1, an HVR-H2 sequence of SEQ ID NO: 2, and an HVR-H3 sequenceof SEQ ID NO: 3. In other aspects, the present disclosure provides achimeric antigen receptor (CAR) that comprises an antibody fragmentcomprising a light chain variable region comprising an HVR-L1 sequenceof SEQ ID NO: 5, an HVR-L2 sequence of SEQ ID NO: 8, and an HVR-L3sequence of SEQ ID NO: 9 and/or a heavy chain variable region comprisingan HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQ ID NO: 2,and an HVR-H3 sequence of SEQ ID NO: 3. In other aspects, the presentdisclosure provides a chimeric antigen receptor (CAR) that specificallybinds to an epitope comprising N-acetylglucosamine orN-acetyl-galactosamine, wherein the CAR comprises a light chain variableregion comprising (a) an HVR-L1 sequence of SEQ ID NO: 4, an HVR-L2sequence of SEQ ID NO: 6, and an HVR-L3 sequence of SEQ ID NO: 9, (b) anHVR-L1 sequence of SEQ ID NO: 21, an HVR-L2 sequence of SEQ ID NO: 22,and an HVR-L3 sequence of SEQ ID NO: 9, (c) an HVR-L1 sequence of SEQ IDNO: 4, an HVR-L2 sequence of SEQ ID NO: 18, and an HVR-L3 sequence ofSEQ ID NO: 9, (d) an HVR-L1 sequence of SEQ ID NO: 5, an HVR-L2 sequenceof SEQ ID NO: 7, and an HVR-L3 sequence of SEQ ID NO: 9, or (e) anHVR-L1 sequence of SEQ ID NO: 5, an HVR-L2 sequence of SEQ ID NO: 8, andan HVR-L3 sequence of SEQ ID NO: 9; and a heavy chain variable regioncomprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3. In other aspects, thepresent disclosure provides a chimeric antigen receptor (CAR) thatspecifically binds to an epitope comprising N-acetylglucosamine orN-acetyl-galactosamine, wherein the CAR comprises a light chain variableregion comprising an HVR-L1 sequence of SEQ ID NO: 5, an HVR-L2 sequenceof FTSX₁LX₂S (SEQ ID NO: 25), and an HVR-L3 sequence of SEQ ID NO: 9,wherein X₁ is T or S and X₂ is Q or E and a heavy chain variable regioncomprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3. In some embodiments,the HVR-L2 sequence comprises the amino acid sequence of SEQ ID NO: 8.In some embodiments, the CAR comprises a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO:26. Insome embodiments, the HVR-L2 sequence comprises the amino acid sequenceof SEQ ID NO: 7. In some embodiments, the CAR comprises a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 12, 13,19, or 23. In some embodiments, the CAR comprises a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 13. In someembodiments of any of the above embodiments, the CAR comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO:10 or SEQ ID NO: 33. In some embodiments, the CAR comprises a lightchain variable region comprising the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:26 and a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 33. In someembodiments, the CAR comprises a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 12 and a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:33. In some embodiments, the CAR comprises a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 23 and a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 10 orSEQ ID NO: 33. In some embodiments, the CAR comprises a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 19 anda heavy chain variable region comprising the amino acid sequence of SEQID NO: 10 or SEQ ID NO: 33. In some embodiments, the CAR comprises alight chain variable region comprising the amino acid sequence of SEQ IDNO: 13 and a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 10 or SEQ ID NO: 33. In some embodiments, thelight chain variable region and the heavy chain variable region arehumanized or human. In some embodiments, the CAR comprises a human CD3zeta endodomain sequence. In some embodiments, the CAR further comprisesa human CD28 endodomain sequence. In some embodiments, the CARcomprises, from N-terminus to C-terminus, the heavy chain variableregion, a linker, the light chain variable region, a CD8 hinge region, ahuman CD28 endodomain, and a human CD3 zeta endodomain. In someembodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 34.In some embodiments, the CAR specifically binds to an epitope comprisingN-acetylglucosamine and an epitope comprising N-acetyl-galactosamine. Insome embodiments, the epitope is expressed on a cell surface of a cancercell. In some embodiments, the cancer cell is selected from the groupconsisting of a pancreatic adenocarcinoma cell, a colon adenocarcinomacell, a rectal adenocarcinoma cell, an esophageal adenocarcinoma cell, aleukemia cell, an adenoid carcinoma cell, a fibrosarcoma cell, aduodenal adenocarcinoma cell, a glioma cell, a hepatocarcinoma cell, alung cancer cell, a breast cancer cell, a glioblastoma cell, an ovariancarcinoma cell, and a cervical adenocarcinoma cell. In some embodiments,the pancreatic adenocarcinoma cell is a pancreatic ductal adenocarcinomacell. In some embodiments, the CAR is expressed on a cell surface of a Tcell, and wherein binding of the CAR to the epitope expressed on thecell surface of the cancer cell leads to killing of the cancer cell bythe T cell.

In other aspects, the present disclosure provides an isolatedpolynucleotide comprising a nucleic acid sequence encoding the CARaccording to any one of the above embodiments. In other aspects, thepresent disclosure provides a vector comprising a nucleic acid sequenceencoding the CAR according to any one of the above embodiments. In someembodiments, the vector is a viral vector. In some embodiments, thevector is a lentiviral vector. In other aspects, the present disclosureprovides an isolated T cell comprising the polynucleotide according toany one of the above embodiments or the vector according to any one ofthe above embodiments. In other aspects, the present disclosure providesan isolated T cell comprising the CAR according to any one of the aboveembodiments, wherein the CAR is expressed on a cell surface of the Tcell. In some embodiments, the T cell is a human T cell.

In other aspects, the present disclosure provides a method for treatingor preventing cancer in an individual, comprising administering to theindividual an effective amount of a composition comprising the T cellaccording to any of the above embodiments. In some embodiments, thecancer is selected from the group consisting of pancreatic cancer,colorectal cancer, esophageal cancer, leukemia, adenocarcinoma,fibrosarcoma, duodenal adenocarcinoma, brain cancer, liver cancer, lungcancer, breast cancer, ovarian cancer, and cervical cancer. In someembodiments, the cancer is selected from the group consisting ofpancreatic cancer, colorectal cancer, lung cancer, and brain cancer. Insome embodiments, the individual is a human. In some embodiments, themethod further comprises administering to the individual an amount ofanother anti-cancer agent, whereby the T cell and the anti-cancer agentin conjunction provide effective treatment or prevention of cancer inthe individual. In some embodiments, the anti-cancer agent is achemotherapeutic agent.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present invention. These and other aspects of theinvention will become apparent to one of skill in the art. These andother embodiments of the invention are further described by the detaileddescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A & 1B show in vitro binding of humanized antibodies VK3 and VK4to N-acetylglucosamine (NAcGlu; FIG. 1A) and N-acetyl-galactosamine(NAcGal; FIG. 1B).

FIG. 2 shows inhibition of antibody VK3 (“K3”) and VK4 (“K4”) binding toN-acetylglucosamine (10 μg) by pre-treatment with N-acetylglucosamine(NAcGlu) or N-acetyl-galactosamine (NAcGal), as compared to nopre-treatment (Ab alone).

FIG. 3 shows the effect of humanized K3 (VK3) and K4 (VK4) antibody onintestinal morphology in a mouse IBD model induced bytrinitrobenzenesulphonic acid (TNBS), as judged by macroscopicassessment conducted on day 7. Treatment groups include G1: untreated(n=5); G2: 200 μg Adalimumab/animal, intraperitoneal (IP) (n=5); G3: 5μg VK3/animal, intraperitoneal (IP) (n=5); and G4: 5 μg VK4/animal,intraperitoneal (IP) (n=5). *indicates p<0.05 by t-test (comparisonswere between the treatment group G3 and the untreated control group G1).

FIG. 4 shows representative gross images taken at day 7 of the IBD modelinduced by TNBS. Treatment with test antibodies was given at 48 hoursafter the induction of the IBD model. Treatment groups are as describedabove for FIG. 3.

FIGS. 5A & 5B show the effects of humanized VK2 and VK3 antibody on pawswelling volume (FIG. 5A) and total body weight (FIG. 5B) in a ratcollagen-induced arthritis model, as compared to saline treatment.Treatment groups include G1: untreated (n=5); G2: antibody VK2 (“K2”) at250 μg/kg once every other day, intravenously (n=5); G3: antibody VK3(“K3”) at 250 μg/each once every other day, intravenously (n=5).

FIGS. 6A & 6B show representative gross images taken at day 5post-dosing (i.e., after 2 doses; FIG. 6A) or day 28 post-dosing (i.e.,after 14 doses; FIG. 6B) of the rat collagen-induced arthritis model.Treatment groups are as described above for FIGS. 5A & 5B.

FIGS. 7A & 7B show the percentage of inhibition that the antibodies VK3(FIG. 7A; “K3”) and VK4 (FIG. 7B; “K4”) demonstrated on cellular growthof a PDX-P3 pancreatic cancer model at various concentrations, asindicated.

FIGS. 8A-8C show the effects of treatment with antibody VK4 (“K4”) ontumor volume (FIG. 8A), tumor growth inhibition (TGI; FIG. 8B), andtumor size (images in FIG. 8C) in the PDX-P3 pancreatic cancer model, ascompared to untreated controls or another antibody (7C).

FIG. 9 shows the percentage of inhibition that the antibody VK3 (“K3”)demonstrated on cellular growth of a PDX-P3 rectal cancer model atvarious concentrations, as indicated.

FIGS. 10A & 10B show the effects of treatment with antibody VK3 (“K3”)or VK4 (“K4”) on tumor volume (FIG. 10A) and tumor growth inhibition(TGI; FIG. 10B) in the PDX-P3 rectal cancer model, as compared tountreated controls.

FIG. 11 shows the reactivity of antibodies VK3 (“K3”) and VK4 (“K4”),compared to a negative control (phosphate buffered saline, PBS) againstvarious human tumor cell lines, as indicated. Cell lines tested include:NCI-H446 (small cell lung cancer), H1299 (non-small cell lungcarcinoma), ECAP-1090 (esophageal adenocarcinoma; Jurkat (acute T cellleukemia), ACC-2 (adenoid cystic carcinoma cells), HT1080(fibrosarcoma), HUTU80 (duodenum adenocarcinoma), MCF-7 (breastcarcinoma) and Hela (cervical cancer).

FIG. 12A shows a vector map of the lentiviral chimeric antigen receptor(CAR) vector used to generate CAR-T cells. FIG. 12B shows expected DNAbands after EcoRI-BamHI digest of the lentiviral vector shown in FIG.12A.

FIGS. 13-16 show CAR-T cell-mediated cell killing of tumor cell lines.Graphs depict percent lysis of target cells vs. indicated E/T ratios.Shown are assays for CAR-T-mediated killing of A549 lung adenocarcinoma(FIG. 13), BxPc-3 pancreatic adenocarcinoma (FIG. 14), HCT116 colorectalcarcinoma (FIG. 15), and U87 glioblastoma (FIG. 16) cell lines.

DETAILED DESCRIPTION

The present disclosure provides, inter alia, new antibodies thatspecifically bind N-acetylglucosamine and N-acetyl galactosamine.Multiple such antibodies are demonstrated herein to treat one or moresymptoms of inflammation, cancer, and/or autoimmune disease in a varietyof in vivo models. In particular, these antibodies were found to haveincreased efficacy as compared to existing antibodies, e.g., inmitigation of inflammation and ulceration in an inflammatory boweldisease (IBD) model, reduction of inflammation in a collagen-inducedarthritis model, and inhibition of tumor growth in a pancreatic orrectal cancer model. In addition, these antibodies were demonstrated tobind to a number of human tumor cell lines representing a range ofdifferent cancer types. Variable domains of an antibody bindingN-acetylglucosamine and N-acetyl galactosamine were also found tomediate cell killing of a wide range of tumor cell lines in the contextof a chimeric antigen receptor expressed by human T cells.

I. General Techniques

The techniques described or referenced herein are well understood andemployed using conventional methodology by those skilled in the art,such as, for example, the widely utilized methodologies described inSambrook et al., Molecular Cloning: A Laboratory Manual 3d edition(2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.,(2003)); Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual,and Animal Cell Culture (R. I. Freshney, ed. (1987)); Methods inMolecular Biology, Humana Press; Monoclonal Antibodies: A PracticalApproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);and Cancer: Principles and Practice of Oncology (V. T. DeVita et al.,eds., J.B. Lippincott Company, 1993).

II. Definitions

Before describing the invention in detail, it is to be understood thatthis invention is not limited to particular compositions or biologicalsystems, which can, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a molecule”optionally includes a combination of two or more such molecules, and thelike.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

The term “inflammation” refers to the complex biological response ofbody tissues to harmful stimuli, such as pathogens, damaged cells, orirritants. The classical signs of acute inflammation include withoutlimitation pain, heat, redness, swelling, and loss of function.Inflammation is a generic response, and therefore it is considered amechanism of innate immunity. Inflammation can be classified as acute orchronic. Acute inflammation refers to the initial response of the bodyto harmful stimuli and is achieved by the increased movement of plasmaand leukocytes (especially granulocytes) from the blood into the injuredtissues. A series of biochemical events propagates and matures theinflammatory response, involving the local vascular system, the immunesystem, and various cells within the injured tissue. Prolongedinflammation, known as chronic inflammation, leads to a progressiveshift in the type of cells present at the site of inflammation and ischaracterized by simultaneous destruction and healing of the tissue fromthe inflammatory process.

The term “inflammatory cells” refers to leukocytes (e.g., neutrophils,macrophages, monocytes, eosinophils, basophils, and lymphocytes) thatnormally reside in the blood and move into the inflamed tissue viaextravasation to aid in inflammation. Some act as phagocytes, ingestingbacteria, viruses, and cellular debris. Others release enzymaticgranules that damage pathogenic invaders. Leukocytes also releaseinflammatory mediators that develop and maintain the inflammatoryresponse. In general, acute inflammation is mediated by granulocytes,whereas chronic inflammation is mediated by mononuclear cells such asmonocytes and lymphocytes. In some embodiments, inflammatory cells mayinclude lymphocytes, such as T- or B-cells.

The term “inflammatory bowel disease (IBD)” refers to the pathologicalstate characterized by chronic inflammation of all or part of digestivetract. IBD primarily includes ulcerative colitis and Crohn's disease.Both usually involve severe diarrhea, pain, fatigue, and weight loss.Ulcerative colitis is a form of IBD that causes long-lastinginflammation and sores (ulcers) in large intestine (colon) and rectum.Crohn's disease is a form of IBD that causes inflammation of thedigestive tract. In Crohn's disease, inflammation often spreads deepinto affected tissues. The inflammation can involve different areas ofthe digestive tract such as the large intestine, small intestine orboth. Collagenous colitis and lymphocytic colitis also are consideredinflammatory bowel diseases but are usually regarded separately fromclassic inflammatory bowel disease.

The terms “cancer” and “cancer cells” refer to or describe thephysiological condition in animals that is typically characterized byunregulated cell growth. Examples of cancers include, but are notlimited to, lung cancer including small cell lung cancer, adenocarcinomaof the lung and squamous carcinoma of the lung, hepatocellular cancer,brain cancer including malignant oligodendroglioma, glioblastoma orglioma, gastrointestinal cancer including but not limited to esophagealcancer, gastric cancer, intestinal cancer, colon cancer and colorectalcancer, kidney clear cell carcinoma, skin basal cell carcinoma, skinsquamous cell carcinoma, throat carcinoma, Hodgkin's lymphoma, thyroidmedullary carcinoma, pancreatic cancer, adenocarcinoma, fibrosarcoma,cervical cancer, ovarian cancer, bladder cancer, cancer of the urinarytract, breast cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, prostate cancer, melanoma, multiple myeloma and B-celllymphoma, leukemias, duodenal cancer (e.g, adenocarcinoma), andassociated metastases. In some embodiments, the type of cancer isselected from: brain cancer, liver cancer, lung cancer, breast cancer,ovarian cancer, and cervical cancer. In some embodiments, the cancercell is selected from: a glioma cell, a hepatocarcinoma cell, a lungcancer cell, a breast cancer cell, an ovarian carcinoma cell, and acervical adenocarcinoma cell. In some embodiments, the type of cancer isselected from: pancreatic cancer, colon cancer, rectal cancer,esophageal cancer, leukemia, adenocarcinoma, fibrosarcoma, duodenaladenocarcinoma, brain cancer, liver cancer, lung cancer, breast cancer,ovarian cancer, and cervical cancer.

The term “immunoglobulin” (Ig) is used interchangeably with “antibody”herein. The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments (e.g., a Fabfragment, scFv, minibody, diabody, scFv multimer, or bispecific antibodyfragment) so long as they exhibit the desired biological activity.

As used herein, the term “specifically binds to” or is “specific for”refers to measurable and reproducible interactions such as bindingbetween a target and an antibody, that is determinative of the presenceof the target in the presence of a heterogeneous population of moleculesincluding biological molecules. For example, an antibody thatspecifically binds to a target (which can be an epitope) is an antibodythat binds this target with greater affinity, avidity, more readily,and/or with greater duration than it binds to other targets. In certainembodiments, an antibody that specifically binds to a target has adissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM.In another embodiment, specific binding can include, but does notrequire exclusive binding.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domains of the heavy chain and light chain may be referred toas “V_(H)” and “V_(L)”, respectively. These domains are generally themost variable parts of the antibody (relative to other antibodies of thesame class) and contain the antigen binding sites.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and defines the specificity of aparticular antibody for its particular antigen. However, the variabilityis not evenly distributed across the entire span of the variabledomains. Instead, it is concentrated in three segments called“hypervariable regions (HVRs)” both in the light-chain and the heavychain variable domains (for a total of 6 HVRs per antibody orantigen-binding fragment thereof). As used herein, a “hypervariableregion (HVR)” contains highly variable sequence that confers specificantigen-binding to an antibody. The more highly conserved portions ofvariable domains are called the framework regions (FR). The HVRs in eachchain are held together in close proximity by the FR regions and, withthe HVRs from the other chain, contribute to the formation of theantigen binding site of antibodies (see Kabat et al., Sequences ofImmunological Interest, Fifth Edition, National Institute of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inthe binding of antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody inantibody-dependent-cellular toxicity.

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variabledomain residues are numbered according to Kabat et al., supra, for eachof these definitions.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations and/orpost-translation modifications (e.g., isomerizations, amidations) thatmay be present in minor amounts. Monoclonal antibodies are highlyspecific, being directed against a single antigenic site. In contrast topolyclonal antibody preparations which typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by a variety of techniques, including, for example, the hybridomamethod (e.g., Kohler and Milstein., Nature, 256:495-97 (1975);recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567);phage-display technologies (see, e.g., Clackson et al., Nature,352:624-628 (1991)); and technologies for producing human or human-likeantibodies in animals that have parts or all of the human immunoglobulinloci or genes encoding human immunoglobulin sequences (see, e.g.,Jakobovits et al., Proc. Nat'l Acad. Sci. USA 90:2551 (1993); Lonberg etal., Nature 368:856-859 (1994)).

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments; minibodies; diabodies; scFvs; scFv multimers; linearantibodies; single-chain antibody molecules; and bispecific ormultispecific antibodies formed from antibody fragments.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the VH and VL antibody domains connected into asingle polypeptide chain. Preferably, the scFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the scFv to form the desired structure for antigen binding. Fora review of the scFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments prepared byconstructing scFv fragments (see preceding paragraph) with short linkers(about 5-10) residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,thereby resulting in a bivalent fragment, i.e., a fragment having twoantigen-binding sites. Diabodies are described in greater detail in, forexample, EP 404,097; WO 93/11161; Hollinger et al., Proc. Nat'l Acad.Sci. USA 90:6444-48 (1993).

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is(are) identical with or homologous to corresponding sequencesin antibodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al., Proc. Nat'l Acad. Sci. USA, 81:6851-55(1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from an HVR of therecipient are replaced by residues from an HVR of a non-human species(donor antibody) such as mouse, rat, rabbit or non-human primate havingthe desired specificity, affinity, and/or capacity. For further details,see, e.g., Jones et al., Nature 321:522-525 (1986).

A “human antibody” is one that possesses an amino-acid sequencecorresponding to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. Human antibodies can be produced using varioustechniques known in the art, such as the methods described in Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985); Boerner et al., J. Immunol., 147(1):86-95 (1991).

An “isolated” antibody is one that has been identified, separated and/orrecovered from a component of its production environment (e.g.,naturally or recombinantly). Preferably, the isolated polypeptide isfree of contaminant components that would typically interfere with usesfor the antibody, e.g., enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes.

As used herein, the term “treatment” refers to clinical interventiondesigned to alter the natural course of the individual or cell beingtreated during the course of clinical pathology. Desirable effects oftreatment include decreasing the rate of disease progression,ameliorating or palliating the disease state, and remission or improvedprognosis. An individual is successfully “treated”, for example, if oneor more symptoms associated with cancer are mitigated or eliminated.

As used herein, the term “prevention” includes providing prophylaxiswith respect to occurrence or recurrence of a disease in an individual.An individual may be predisposed to, susceptible to a type of cancer, orat risk of developing a type of cancer, but has not yet been diagnosedwith the disease.

An “effective amount” refers to at least an amount effective, at dosagesand for periods of time necessary, to achieve the desired therapeutic orprophylactic result. An effective amount can be provided in one or moreadministrations.

A “therapeutically effective amount” is at least the minimumconcentration required to effect a measurable improvement of aparticular disorder (e.g., cancer). A therapeutically effective amountherein may vary according to factors such as the disease state, age,sex, and weight of the patient, and the ability of the monoclonalantibody to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the monoclonal antibody are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at the dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typicallybut not necessarily, since a prophylactic dose is used in subjects priorto or at an earlier stage of disease, a prophylactically effectiveamount may be less than a therapeutically effective amount.

As used herein, administration “in conjunction” with another compound orcomposition includes simultaneous administration and/or administrationat different times. Administration in conjunction also encompassesadministration as a co-formulation or administration as separatecompositions, including at different dosing frequencies or intervals,and using the same route of administration or different routes ofadministration.

An “individual” for purposes of treatment or prevention refers to anyanimal classified as a mammal, including humans, domestic and farmanimals, and zoo, sport, or pet animals, such as dogs, horses, rabbits,cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, and thelike. In some embodiments, the individual is human. In some embodiments,the individual is a non-human animal.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; low molecular weight (lessthan about 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; amino acids such as glycine, glutamine,asparagine, arginine or lysine; carbohydrates including glucose,mannose, or dextrins; and/or nonionic surfactants such as TWEEN™,polyethylene glycol (PEG), and PLURONICS™.

“Pharmaceutically acceptable” buffers and salts include those derivedfrom both acid and base addition salts of the above indicated acids andbases. Specific buffers and/or salts include histidine, succinate andacetate.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase or by a syntheticreaction.

An “isolated” polynucleotide encoding the antibodies herein is a nucleicacid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the environment in which it was produced. The isolated nucleic acidmolecules encoding the polypeptides and antibodies herein are in a formother than in the form or setting in which it is found in nature.Preferably, the isolated nucleic acid is free of association with allcomponents associated with the production environment.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. Types of vectors include plasmids (i.e., circulardouble stranded DNA into which additional DNA segments may be ligated)and viral vectors. Certain vectors are capable of autonomous replicationin a host cell into which they are introduced (e.g., bacterial vectorshaving a bacterial origin of replication and episomal mammalianvectors). Other vectors can be integrated into the genome of a host celland replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors,” or simply, “expression vectors.” In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

III. Saccharides

Certain aspects of the present disclosure are related to epitopescontaining saccharides. As used herein, a “saccharide” may refer to amonosaccharide, an oligosaccharide or a polysaccharide. Monosaccharidesinclude but not limited to fructose, glucose, mannose, fucose, xylose,galactose, lactose, N-acetylneuraminic acid, N-acetyl-galactosamine,N-acetylglucosamine, and sialic acids. An oligosaccharide is asaccharide polymer containing multiple sugar monomers linked byglycosidic linkages of component sugars.

Glycoproteins or proteosaccharides refer to proteins linked withsaccharides and may typically contain, for example, O- or N-glycosidiclinkages of monosaccharides to compatible amino acid side chains inproteins or to lipid moieties. As used herein, the terms “glycan” and“glycosyl moiety” may be used interchangeably to refer to a saccharidealone or a sugar as the saccharide component of a glycoprotein. Twotypes of glycosylation are known in the art: N-linked glycosylation tothe amide nitrogen of asparagine side chains and O-linked glycosylationto the hydroxy oxygen of serine and threonine side chains. Othersaccharides include but not limited to O-GlcNAc, GAG Chain,glycosaminosaccharides, and glycosphinglipid. O- and N-linkedsaccharides are very common in eukaryotes but may also be found,although less commonly, in prokaryotes.

Certain aspects of the present disclosure relate to N-acetylglucosamineand N-acetyl-galactosamine. N-acetylglucosamine may refer to any aminosugar compound that includes an N-linked glucosamine moiety. As usedherein, N-acetylglucosamine may refer to the monosaccharide on its own,or the monosaccharide as a component of a larger polysaccharide. As usedherein, N-acetylglucosamine may refer to a saccharide entity on its own,or the saccharide as the glycan component of a glycoprotein or proteinglycosylated with one or more N-acetylglucosamine-based components(e.g., mono- or poly-saccharides that contain N-acetylglucosamine).

N-acetyl-galactosamine may refer to any compound that includesglucosamine N-linked to an acetic acid moiety. N-acetyl-galactosaminemay refer to any amino sugar compound that includes an N-linkedgalactosamine moiety. As used herein, N-acetyl-galactosamine may referto the monosaccharide on its own, or the monosaccharide as a componentof a larger polysaccharide. As used herein, N-acetyl-galactosamine mayrefer to a saccharide entity on its own, or the saccharide as the glycancomponent of a glycoprotein or protein glycosylated with one or moreN-acetyl-galactosamine moieties (e.g., mono- or poly-saccharides thatcontain N-acetyl-galactosamine).

While many proteins are known to be glycosylated, glycoproteins areoften found on the exterior surface of cells (i.e., extracellular) orsecreted. Because of this, glycoproteins are highly accessible toexternal agents (e.g., exogenous compounds administered to a patient).For example, components that specifically recognize certainglycoproteins (e.g., antibodies or lectins) are able to bind, in anintact organism, to cells that express these glycoproteins on their cellsurface. Components that specifically recognize certain glycoproteinsare also able to bind secreted saccharides or glycoproteins, for examplethose that may be found freely in certain tissue samples (including,without limitation, in blood or serum).

Lectins are known in the art as sugar-binding proteins which are able torecognize cognate sugar moieties with high specificity. These highlyspecific binding interactions may be exploited, for example, for thedetection of specific saccharides in tissues (e.g., for the detection ofcell surface proteins modified by glycosylation with specific sugarmoieties). Lectins may include, for example, animal lectins, plantlectins, and pathogen lectins. In mammals, lectins are known to playimportant roles in the immune system by, e.g., recognizing carbohydratesthat are found exclusively on pathogens, or that are inaccessible onhost cells.

Certain aspects of the present disclosure use plant lectins to detectthe presence or expression of specific sugar moieties. For example,plant lectins may include but not limited to lectins specific tofructose, mannose, glucose, fucose, galactose, N-acetyl-galactosamine,and N-acetyl-glucosamine.

IV. Antibodies

Epitope Binding

Certain aspects of the present disclosure relate to antibodies thatspecifically bind to an epitope containing N-acetylglucosamine orN-acetyl-galactosamine. As described above, such antibodies will displaymeasurable and reproducible interactions such as binding to an epitopecontaining N-acetylglucosamine or N-acetyl-galactosamine. For example,an antibody that specifically binds to an epitope is an antibody thatbinds this epitope with greater affinity, avidity, more readily, and/orwith greater duration than it binds to other targets. Examples ofepitopes containing N-acetylglucosamine or N-acetyl-galactosamineinclude glycoproteins containing N-acetylglucosamine orN-acetyl-galactosamine glycans, for example and without limitation cellsurface glycoproteins bearing an N-acetylglucosamine orN-acetyl-galactosamine moiety expressed on the surface of a cancer cell.

Specific binding can include, but does not require exclusive binding.While antibodies that specifically bind to an epitope containingN-acetylglucosamine or N-acetyl-galactosamine, they may also be found tobind to other epitopes not containing these moieties, e.g., with alesser binding affinity than epitopes containing N-acetylglucosamine orN-acetyl-galactosamine.

In some embodiments, antibodies specifically bind to epitopes containingN-acetylglucosamine and N-acetyl-galactosamine. For example, an antibodymay be capable of specific binding to an epitope containingN-acetylglucosamine and to an epitope containing N-acetyl-galactosamine.In some embodiments, an antibody may be capable of specific binding toan epitope containing both N-acetylglucosamine andN-acetyl-galactosamine.

In some embodiments, the binding of the antibody to the epitopecontaining N-acetylglucosamine and/or N-acetyl-galactosamine expressedon the cell surface of the cancer cell inhibits growth of the cancercell. As used herein, inhibiting the growth of a cell may refer toinhibition its rate of proliferation. Without wishing to be bound totheory, through binding to the cell surface, antibodies may inhibit thegrowth of cells by a variety of mechanisms. For example, antibodybinding to the cell surface may be toxic to the cell or otherwise causecell death, for example and without limitation, apoptosis or necrosis.Antibody binding to the cell surface may slow or stop cellproliferation. Antibody binding to the cell surface glycoprotein on thecell surface may inhibit or enhance a function of the glycoprotein, forexample a cell signaling function, and in so doing the antibody bindingmay inhibit the growth of the cell. Antibody binding to the cell surfacemay compete with an extrinsic ligand that accelerates the growth of thecell through binding to the cell surface, for example a growth factor.This competition may be indirect, i.e., the antibody need notcompetitively bind an epitope on the same glycoprotein as the extrinsicligand. Antibody binding to the cell surface may also attract one ormore components of the immune system, such as natural killer or NKcells, that inhibit the growth of antibody-bound cells. The mechanism(s)by which different antibodies inhibit the growth of cells throughbinding epitopes on the cell surface may be different depending on thecellular context or the specific antibody or epitope.

Antibody Features

Certain antibodies that specifically bind to an epitope containingN-acetylglucosamine or N-acetyl-galactosamine are described andcharacterized in the present disclosure. In some embodiments, theantibody is 1C5C9, e.g., as described in PCT/CN2015/087717. In someembodiments, the antibody is a humanized form of antibody 1C5C9, e.g.,as described in PCT/CN2015/087717. For example, in some embodiments, theantibody is derived from a humanized parental 1C5C9 antibody. In someembodiments, the humanized parental 105C9 antibody comprises a heavychain variable region comprising an HVR-H1 sequence of YTFPDYNIH (SEQ IDNO: 1), an HVR-H2 sequence of CIYPYNGNTA (SEQ ID NO: 2), and an HVR-H3sequence of SDLYYFGSRGFD (SEQ ID NO: 3). In some embodiments, thehumanized parental 105C9 antibody comprises a light chain variableregion comprising an HVR-L1 sequence of RASQDISTYLN (SEQ ID NO: 4), anHVR-L2 sequence of FTSRLHS (SEQ ID NO: 6), and an HVR-L3 sequence ofQQGNTLPW (SEQ ID NO: 9). In some embodiments, the humanized parental105C9 antibody comprises a heavy chain variable region comprising theamino acid sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFPDYNIHWVRQAPGQGLEWMGCIYPYNGNTAYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSDLYYFGSRGFDY WGQGTLVTVSSA(SEQ ID NO: 10), and/or a light chain variable region comprising theamino acid sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDISTYLNWYQQKPGKAPKWYFTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGNTLPWTFGGGTKLEIK (SEQ ID NO: 12).

In some embodiments, the humanized parental 105C9 antibody comprises aheavy chain comprising the amino acid sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFPDYNIHWVRQAPGQGLEWMGCIYPYNGNTAYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSDLYYFGSRGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11);and/or a light chain comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDISTYLNWYQQKPGKAPKWYFTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGNTLPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 15).

In some embodiments, the antibody is a humanized form of antibody 105C9.In some embodiments, the antibody comprises a light chain variableregion comprising an HVR-L1 sequence of SEQ ID NO: 5, an HVR-L2 sequenceof FTSX₁LX₂S (SEQ ID NO: 25), and an HVR-L3 sequence of SEQ ID NO: 9,where X₁ is T or S and X₂ is Q or E.

In some embodiments, the antibody is humanized 1C5-VK1. In someembodiments, the antibody comprises a heavy chain variable regioncomprising an HVR-H1 sequence of YTFPDYNIH (SEQ ID NO: 1), an HVR-H2sequence of CIYPYNGNTA (SEQ ID NO: 2), and an HVR-H3 sequence ofSDLYYFGSRGFD (SEQ ID NO: 3). In some embodiments, the antibody comprisesa light chain variable region comprising an HVR-L1 sequence ofQASQDISTYLN (SEQ ID NO: 21), an HVR-L2 sequence of FTSNLET (SEQ ID NO:22), and an HVR-L3 sequence of QQGNTLPW (SEQ ID NO: 9).

In some embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFPDYNIHWVRQAPGQGLEWMGCIYPYNGNTAYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSDLYYFGSRGFDY WGQGTLVTVSSA(SEQ ID NO: 10), and/or a light chain variable region comprising theamino acid sequence ofDIQMTQSPSSLSASVGDRVTITCQASQDISTYLNWYQQKPGKAPKLLIYFTSNLETGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGNTLPWTFGGGTKLEIK (SEQ ID NO: 23).

In some embodiments, the antibody comprises a heavy chain comprising theamino acid sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFPDYNIHWVRQAPGQGLEWMGCIYPYNGNTAYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSDLYYFGSRGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11),and/or a light chain comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITCQASQDISTYLNWYQQKPGKAPKLLIYFTSNLETGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGNTLPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 24).

In some embodiments, the antibody is humanized 1C5-VK2. In someembodiments, the antibody comprises a heavy chain variable regioncomprising an HVR-H1 sequence of YTFPDYNIH (SEQ ID NO: 1), an HVR-H2sequence of CIYPYNGNTA (SEQ ID NO: 2), and an HVR-H3 sequence ofSDLYYFGSRGFD (SEQ ID NO: 3). In some embodiments, the antibody comprisesa light chain variable region comprising an HVR-L1 sequence ofRASQDISTYLN (SEQ ID NO: 4), an HVR-L2 sequence of FTSSLQS (SEQ ID NO:18), and an HVR-L3 sequence of QQGNTLPW (SEQ ID NO: 9).

In some embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFPDYNIHWVRQAPGQGLEWMGCIYPYNGNTAYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSDLYYFGSRGFDY WGQGTLVTVSSA(SEQ ID NO: 10), and/or a light chain variable region comprising theamino acid sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDISTYLNWYQQKPGKAPKWYFTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGNTLPWTFGGGTKLEIK (SEQ ID NO: 19).

In some embodiments, the antibody comprises a heavy chain comprising theamino acid sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFPDYNIHWVRQAPGQGLEWMGCIYPYNGNTAYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSDLYYFGSRGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11),and/or a light chain comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDISTYLNWYQQKPGKAPKWYFTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGNTLPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 20).

In some embodiments, the antibody is humanized 1C5-VK3. In someembodiments, the antibody comprises a heavy chain variable regioncomprising an HVR-H1 sequence of YTFPDYNIH (SEQ ID NO: 1), an HVR-H2sequence of CIYPYNGNTA (SEQ ID NO: 2), and an HVR-H3 sequence ofSDLYYFGSRGFD (SEQ ID NO: 3). In some embodiments, the antibody comprisesa light chain variable region comprising an HVR-L1 sequence ofRASQDISTYLA (SEQ ID NO: 5), an HVR-L2 sequence of FTSTLQS (SEQ ID NO:7), and an HVR-L3 sequence of QQGNTLPW (SEQ ID NO: 9).

In some embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFPDYNIHWVRQAPGQGLEWMGCIYPYNGNTAYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSDLYYFGSRGFDY WGQGTLVTVSSA(SEQ ID NO: 10), and/or a light chain variable region comprising theamino acid sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDISTYLAWYQQKPGKAPKLLIYFTSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQQGNTLPWTFGGGTKLEIK (SEQ ID NO: 13).

In some embodiments, the antibody comprises a heavy chain comprising theamino acid sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFPDYNIHWVRQAPGQGLEWMGCIYPYNGNTAYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSDLYYFGSRGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11),and/or a light chain comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDISTYLAWYQQKPGKAPKLLIYFTSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQQGNTLPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 16).

In some embodiments, the antibody is humanized 1C5-VK4. In someembodiments, the antibody comprises a heavy chain variable regioncomprising an HVR-H1 sequence of YTFPDYNIH (SEQ ID NO: 1), an HVR-H2sequence of CIYPYNGNTA (SEQ ID NO: 2), and an HVR-H3 sequence ofSDLYYFGSRGFD (SEQ ID NO: 3). In some embodiments, the antibody comprisesa light chain variable region comprising an HVR-L1 sequence ofRASQDISTYLA (SEQ ID NO: 5), an HVR-L2 sequence of FTSSLES (SEQ ID NO:8), and an HVR-L3 sequence of QQGNTLPW (SEQ ID NO: 9).

In some embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFPDYNIHWVRQAPGQGLEWMGCIYPYNGNTAYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSDLYYFGSRGFDY WGQGTLVTVSSA(SEQ ID NO: 10), and/or a light chain variable region comprising theamino acid sequence ofAIQLTQSPSSLSASVGDRVTITCRASQDISTYLAWYQQKPGKAPKLLIYFTSSLESGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKLEIK (SEQ ID NO: 14).

In some embodiments, the antibody comprises a heavy chain comprising theamino acid sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFPDYNIHWVRQAPGQGLEWMGCIYPYNGNTAYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSDLYYFGSRGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11),and/or a light chain comprising the amino acid sequence ofAIQLTQSPSSLSASVGDRVTITCRASQDISTYLAWYQQKPGKAPKLLIYFTSSLESGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 17).

In some embodiments, the antibody comprises a heavy chain variableregion comprising three HVRs of SEQ ID NO: 10 and/or a light chainvariable region comprising three HVRs of SEQ ID NO: 12. In someembodiments, the antibody comprises a heavy chain variable regioncomprising three HVRs of SEQ ID NO: 10 and/or a light chain variableregion comprising three HVRs of SEQ ID NO: 13. In some embodiments, theantibody comprises a heavy chain variable region comprising three HVRsof SEQ ID NO: 10 and/or a light chain variable region comprising threeHVRs of SEQ ID NO: 14. In some embodiments, the antibody comprises aheavy chain variable region comprising three HVRs of SEQ ID NO: 10and/or a light chain variable region comprising three HVRs of SEQ ID NO:19. In some embodiments, the antibody comprises a heavy chain variableregion comprising three HVRs of SEQ ID NO: 10 and/or a light chainvariable region comprising three HVRs of SEQ ID NO: 23. In someembodiments, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 10 and/or a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 12. Insome embodiments, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 10 and/or a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 13. Insome embodiments, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 10 and/or a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 14. Insome embodiments, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 10 and/or a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 19. Insome embodiments, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 10 and/or a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 23

The antibodies useful in the present invention can encompass monoclonalantibodies, polyclonal antibodies, antibody fragments (e.g., Fab,Fab′-SH, Fv, scFv, and F(ab′)₂), chimeric antibodies, bispecificantibodies, multivalent antibodies, heteroconjugate antibodies, fusionproteins comprising an antibody portion, humanized antibodies, and anyother modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site of the required specificity (e.g.,for epitope containing N-acetylglucosamine or N-acetyl-galactosamine),including glycosylation variants of antibodies, amino acid sequencevariants of antibodies, and covalently modified antibodies. Theantibodies may be murine, rat, human, or of any other origin (includingchimeric or humanized antibodies).

In some embodiments, the antibody that specifically binds to an epitopecontaining N-acetylglucosamine or N-acetyl-galactosamine is a monoclonalantibody. For example, the monoclonal antibodies may be made using thehybridoma method first described by Kohler et al., Nature, 256:495(1975), or may be made by recombinant DNA methods (U.S. Pat. No.4,816,567).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen (e.g.,an epitope containing N-acetylglucosamine or N-acetyl-galactosamine).Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (MA) or enzyme-linkedimmunosorbent assay (ELISA).

Monoclonal antibodies may also be made by recombinant DNA methods, suchas those described above. DNA encoding the monoclonal antibodies isreadily isolated and sequenced using conventional procedures, placedinto expression vectors, and transfected into host cells such as E. colicells or CHO cells to produce recombinant monoclonal antibodies.

In some embodiments, the antibody that specifically binds to an epitopecontaining N-acetylglucosamine or N-acetyl-galactosamine is a humanizedantibody. Humanized antibodies include human immunoglobulins (recipientantibody) in which residues from a complementarity determining region(CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. See Jones et al., Nature321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988) andPresta, Curr. Opin. Struct. Biol. 2: 593-596 (1992).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. Humanization can beessentially performed following the method of Jones et al., Nature321:522-525 (1986); or through substituting non-human CDR sequences forthe corresponding sequences of a human antibody. To ensure humanizedantibodies retain high affinity for the antigen, humanized antibodiesmay be prepared by a process of analyzing the parental sequences andvarious conceptual humanized products using three-dimensional models ofthe parental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. In general, the CDR residues are directly and most substantiallyinvolved in influencing antigen binding.

In some embodiments, the antibody that specifically binds to an epitopecontaining N-acetylglucosamine or N-acetyl-galactosamine is a humanantibody. Methods known in the art for producing human antibodiesinclude, without limitation, phage display technology and use oftransgenic animals that produce human antibodies in response to antigen.

In some embodiments, the antibody that specifically binds to an epitopecontaining N-acetylglucosamine or N-acetyl-galactosamine is a chimericantibody. Chimeric antibodies may refer to an antibody in which residuesfrom a complementarity determining region (CDR) or variable regionderived from one species are joined with sequences corresponding to theconstant region from another species. Methods for generating chimericantibodies are known in the art (see, e.g., U.S. Pat. No. 4,816,567).

In some embodiments, the antibody that specifically binds to an epitopecontaining N-acetylglucosamine or N-acetyl-galactosamine is an antibodyfragment. In some embodiments, the antibody that specifically binds toan epitope containing N-acetylglucosamine or N-acetyl-galactosamine is aFab fragment, scFv, minibody, diabody, scFv multimer, or bispecificantibody fragment. Fab, Fv and scFv antibody fragments can all beexpressed in and secreted from E. coli, thus allowing thestraightforward production of large amounts of these fragments, orisolated from phage libraries. Such linear antibody fragments may bemonospecific or bispecific.

In some embodiments, the antibody that specifically binds to an epitopecontaining N-acetylglucosamine or N-acetyl-galactosamine is a bispecificantibody. Bispecific antibodies (BsAbs) are antibodies that have bindingspecificities for at least two different epitopes, including those onthe same or another protein. Methods for making bispecific antibodiesare known in the art. Traditional production of full length bispecificantibodies is based on the coexpression of two immunoglobulinheavy-chain/light chain pairs, where the two chains have differentspecificities.

In some embodiments, the antibody that specifically binds to an epitopecontaining N-acetylglucosamine or N-acetyl-galactosamine is amultivalent antibody. Multivalent antibodies may refer to any antibodywith more than 2 antigen-binding sites. In some embodiments, theantibody that specifically binds to an epitope containingN-acetylglucosamine or N-acetyl-galactosamine is a heteroconjugateantibody. Heteroconjugate antibodies may refer to any antibody createdby linking two antibodies with different specificities, such as by acovalent linkage.

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. DNAs encoding theimmunoglobulin heavy chain fusions and, if desired, the immunoglobulinlight chain, are inserted into separate expression vectors, and areco-transfected into a suitable host organism.

In some embodiments, compositions containing an antibody thatspecifically binds to an epitope containing N-acetylglucosamine orN-acetyl-galactosamine may include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed as part of apharmaceutical composition. Often the physiologically acceptable carrieris an aqueous pH buffered solution. Examples of physiologicallyacceptable carriers include buffers such as phosphate, citrate, andother organic acids; antioxidants including ascorbic acid; low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

Polynucleotides, Vectors Encoding Antibodies, and Host Cells

Certain aspects of the present disclosure relate to the production ofantibodies that specifically bind to an epitope containingN-acetylglucosamine or N-acetyl-galactosamine. In particular, certainaspects relate to isolated polynucleotides containing a nucleic acidsequence encoding an antibody that specifically binds to an epitopecontaining N-acetylglucosamine or N-acetyl-galactosamine. As describedabove, polynucleotides may refer to deoxyribonucleotides,ribonucleotides, modified nucleotides or bases, and/or their analogs.These polynucleotides may be produced in vivo in a host cell or throughin vitro transcription. Polynucleotides encoding an antibody may referto polynucleotides bearing the sequence encoding the antibody as it wasidentified in a cell producing the antibody (e.g., a B cell orhybridoma), or polynucleotides containing synonymous mutations in thesequence that distinguish them from their naturally occurringcounterparts but, due to the inherent degeneracy of the genetic code,encode a similar protein. Polynucleotides may be isolated by any meansknown in the art, including PCR followed by precipitation-basedpurification of the PCR reaction, or a slice of agarose gel containingthe PCR product, or by purification of a vector containing thepolynucleotide from a host cell (e.g., plasmid preparation from E.coli).

Certain aspects of the present disclosure relate to vectors containing anucleic acid sequence encoding an antibody that specifically binds to anepitope containing N-acetylglucosamine or N-acetyl-galactosamine. Forrecombinant production of antibodies or fragments thereof, nucleic acidsencoding the desired antibodies or antibody fragments are isolated andinserted into a replicable vector for further cloning (amplification ofthe DNA) or for expression. DNA encoding the polyclonal or monoclonalantibodies is readily isolated (e.g., with oligonucleotide probes thatspecifically bind to genes encoding the heavy and light chains of theantibody) and sequenced using conventional procedures. Many cloningand/or expression vectors are commercially available.

Vector components generally include, but are not limited to, one or moreof the following, a signal sequence, an origin of replication, one ormore marker genes, a multiple cloning site containing recognitionsequences for numerous restriction endonucleases, an enhancer element, apromoter, and a transcription termination sequence. Both expression andcloning vectors contain a nucleic acid sequence that enables the vectorto replicate in one or more selected host-cells. Such sequences are wellknown for a variety of bacteria, yeast, and viruses. Expression andcloning vectors may also contain a selection gene, known as a selectablemarker, whose expression confers resistance to antibiotics or othertoxins, complements auxotrophic deficiencies, or supplies criticalnutrients not available from complex media.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid encoding the antibodies (e.g., antibodies that specifically bind toan epitope containing N-acetylglucosamine or N-acetyl-galactosamine) orfragments thereof. Promoters suitable for use with prokaryotic hostsinclude the phoA promoter, lactamase and lactose promoter systems,alkaline phosphatase promoter, a tryptophan promoter system, and hybridpromoters such as the tac promoter, although other known bacterialpromoters are also suitable. Promoters for use in bacterial systems alsowill contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding the antibodies and antibody fragments. Promoter sequences areknown for eukaryotes, including the yeast promoters for3-phosphoglycerate kinase or other glycolytic enzymes and mammalianpromoters obtained from the genomes of viruses such as polyoma virus,cytomegalovirus, and most preferably Simian Virus 40 (SV40). Variousheterologous mammalian promoters, e.g., the actin promoter,immunoglobulin promoter, and heat-shock promoters, are also known.Expression vectors used in eukaryotic host cells will also containsequences necessary for the termination of transcription and forstabilizing the mRNA.

Certain aspects of the present disclosure relate to isolated host cellswith vectors containing a nucleic acid sequence encoding an antibodythat specifically binds to an epitope containing N-acetylglucosamine orN-acetyl-galactosamine. Suitable host-cells for cloning or expressingthe DNA encoding antibodies (e.g., antibodies that specifically bind toan epitope containing N-acetylglucosamine or N-acetyl-galactosamine) orfragments thereof in the vectors described herein prokaryotes such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. In addition to prokaryotes,eukaryotic microbes such as filamentous fungi or yeast are also suitablecloning or expression hosts, such as Saccharomyces cerevisiae. For areview discussing the use of yeasts and filamentous fungi for theproduction of therapeutic proteins, see, e.g., Gerngross, Nat. Biotech.22: 1409-1414 (2004). Suitable host-cells for the expression ofglycosylated antibodies or antibody fragments are derived frommulticellular organisms. Examples of invertebrate cells include plantand insect-cells such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Drosophila melanogaster (fruitfly), or Bombyx mori(moth) cells. Examples of useful mammalian host-cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Nat'l Acad. Sci. USA 77:4216(1980)); African green monkey kidney cells (VERO-76, ATCC CRL-1587);human cervical carcinoma cells (HELA, ATCC CCL 2); and a human hepatomaline (Hep G2). For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 255-268. These examples are illustrative rather thanlimiting.

Antibody Production and Purification

Certain aspects of the present disclosure relate to methods of producingan antibody by culturing host cells with vectors containing a nucleicacid sequence encoding an antibody and recovering the antibody from thecell culture. Host cells are transformed with the above-describedexpression or cloning vectors for antibody or antibody fragmentproduction and cultured in conventional nutrient media modified asappropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences.

The host-cells used to produce the antibodies (e.g., antibodies thatspecifically bind to an epitope containing N-acetylglucosamine orN-acetyl-galactosamine) or antibody fragments described herein may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host-cells. Any of these media may besupplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers,nucleotides, antibiotics, trace elements, and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host-cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

When using recombinant techniques, the antibodies (e.g., antibodies thatspecifically bind to an epitope containing N-acetylglucosamine orN-acetyl-galactosamine) or antibody fragments can be producedintracellularly, in the periplasmic space, or secreted directly into themedium. Antibodies prepared from such cells can be purified using, forexample, hydroxylapatite chromatography, gel electrophoresis, dialysis,and affinity chromatography, such as that using protein A or protein Gattached to a matrix (e.g., agarose).

In general, various methodologies for purifying preparing antibodies foruse in research, testing, and clinical applications are well-establishedin the art, consistent with the above-described methodologies and/or asdeemed appropriate by one skilled in the art for a particular antibodyof interest.

V. Cancer

Certain aspects of the present disclosure relate to methods for treatingor preventing cancer in an individual by administering to the individualan effective amount of a composition containing an antibody thatspecifically binds to an epitope containing N-acetylglucosamine orN-acetyl-galactosamine, where the epitope is expressed by a cancer cell.In some embodiments, the epitope is expressed on a cell surface of acancer cell. In some embodiments, binding of the antibody to the epitopeexpressed on the cell surface of the cancer cell inhibits growth of thecancer cell.

Certain glycoproteins, particularly N-acetylglucosamine and/orN-acetyl-galactosamine, have been found to be highly expressed on thecell surface of many types of human cancer cells, as compared to littleor no expression on the cell surface of normal human cells (see, e.g.,PCT/CN2015/087717). As a result, these sugar moieties may serve asbiomarkers for the presence of cancer that may also be used topreferentially target therapeutic agents (e.g., antibodies) to cancercells. Advantageously, antibodies specific to N-acetylglucosamine and/orN-acetyl-galactosamine may bind to the cell surface and are able toinhibit the growth of cancer cells expressing these sugar moieties.

Described herein are novel antibodies that specifically bind to anepitope containing N-acetylglucosamine and/or N-acetyl-galactosamine. Asdescribed below, these antibodies are effective in inhibiting the growthof different types of cancer cells in various tumor xenograft models.Tumor xenograft models are known in the art as a powerful tool fortesting and predicting the drug response of human tumors (see, e.g.,Richmond, A. and Su, Y. (2008) Dis. Model Mech. 1(2-3): 78-82). Theseantibodies are also reactive against a number of different human tumorcell lines. In addition, these antibodies were found to be better thanexisting antibodies in multiple models of inflammation, e.g., IBD andarthritis.

While the results described herein predict that antibodies thatspecifically bind to an epitope containing N-acetylglucosamine orN-acetyl-galactosamine are broadly effective against multiple forms ofhuman cancer. In some embodiments, the cancer cell whose growth isinhibited by the antibody is selected from: a pancreatic adenocarcinomacell, a colon adenocarcinoma cell, a rectal adenocarcinoma cell, anesophageal adenocarcinoma cell, a leukemia cell, an adenoid carcinomacell, a fibrosarcoma cell, a duodenal adenocarcinoma cell, a gliomacell, a hepatocarcinoma cell, a lung cancer cell, a breast cancer cell,a glioblastoma cell, an ovarian carcinoma cell, a cervicaladenocarcinoma cell, a colon carcinoma cell, a stomach or gastriccarcinoma cell, an esophageal carcinoma cell, and a fibrosarcoma cell.

In some embodiments, the cancer cell is a pancreatic adenocarcinomacell. Pancreatic adenocarcinoma cells may refer to any adenocarcinomacells originating from the pancreas, including primary tumors orpancreatic adenocarcinoma cells that have metastasized to other sites.In some embodiments, the pancreatic adenocarcinoma cell is a pancreaticductal adenocarcinoma cell.

In some embodiments, the cancer cell is a colon or rectal adenocarcinomacell. Colon or rectal adenocarcinoma cells may refer to anyadenocarcinoma cells originating from the colon or rectum, includingprimary tumors or adenocarcinoma cells that have metastasized to othersites. In some embodiments, the adenocarcinoma cell is a moderatelydifferentiated colon adenocarcinoma cell.

In some embodiments, the cancer cell is a glioma or glioblastoma cell.Glioma cells may refer to any malignant cell originating from a glialcell, e.g., glial cells of the brain or spine, including primary tumorsor glioma cells that have metastasized to other sites. Glioma cells mayrefer to a homogeneous population of glioma cells, or a mixed populationof cells arising from different types of glia. In some embodiments, theglioma cell may be an oligodendroglioma cell, a brainstem glioma cell,an ependymoma cell, or an optic nerve glioma cell. Gliomas can includegrade III astrocytomas, oligodendrogliomas, and oligoastrocytomas.Glioblastoma cells refer to malignant cells originating from anastrocyte (typically used to characterize grade IV gliomas orastrocytomas), e.g., astrocytes of the brain, including primary tumorsor glioblastoma cells that have metastasized to other sites.Glioblastoma cells may include primary or de novo tumors and secondarytumors and may include other cell types and blood vessels.

In some embodiments, the cancer cell is a hepatocarcinoma cell.Hepatocarcinoma cells may refer to any carcinoma cell originating fromthe liver, including primary tumors or hepatocarcinoma cells that havemetastasized to other sites.

In some embodiments, the cancer cell is a lung cancer cell. Lung cancercells may refer to any cancer cell originating from the lung, includingprimary tumors or lung cancer cells that have metastasized to othersites. In some embodiments, the lung cancer cell may be a non-small-celllung cancer cell. In some embodiments, the lung cancer cell may be alung adenocarcinoma cell. In some embodiments, the lung cancer cell maybe a lung squamous cell carcinoma. In some embodiments, the lung cancercell may be a small-cell lung cancer cell.

In some embodiments, the cancer cell is a breast cancer cell. Breastcancer cells may refer to any cancer cell originating from the breast,including primary tumors or breast cancer cells that have metastasizedto other sites. In some embodiments, the breast cancer cell may be aductal carcinoma in situ cell. In some embodiments, the breast cancercell may be an invasive ductal carcinoma cell. In some embodiments, thebreast cancer cell may be an invasive lobular carcinoma cell.

In some embodiments, the cancer cell is an ovarian carcinoma cell.Ovarian carcinoma cells may refer to any carcinoma cell originating fromthe ovary, including primary tumors or ovarian carcinoma cells that havemetastasized to other sites. In some embodiments, the ovarian carcinomacell may be a surface epithelial-stromal tumor cell. In someembodiments, the ovarian carcinoma cell may be a sex cord-stromal tumorcell. In some embodiments, the ovarian carcinoma cell may be a germ celltumor cell. Ovarian carcinoma cells may refer to a homogeneouspopulation of ovarian carcinoma cells, or a mixed population of cellsarising from different types of ovarian carcinomas.

In some embodiments, the cancer cell is a cervical adenocarcinoma cell.Cervical adenocarcinoma carcinoma cells may refer to any adenocarcinomacell originating from the cervix, including primary tumors or cervicaladenocarcinoma cells that have metastasized to other sites. In someembodiments, the cervical adenocarcinoma cell is an adenosquamouscarcinoma cell.

In some embodiments, the cancer cell is a colon carcinoma cell. Coloncarcinoma cells may refer to any carcinoma cell originating from thecolon or rectum, including primary tumors or colon carcinoma cells thathave metastasized to other sites. In some embodiments, the coloncarcinoma cell is an adenocarcinoma cell. In some embodiments, the coloncarcinoma cell is an adenosquamous carcinoma cell.

In some embodiments, the cancer cell is a stomach or gastric carcinomacell. Stomach or gastric carcinoma cells may refer to any carcinoma celloriginating from the stomach, including primary tumors or stomachcarcinoma cells that have metastasized to other sites. In someembodiments, the stomach or gastric carcinoma cell is an adenocarcinomacell. In some embodiments, the stomach or gastric carcinoma cell is adiffuse type adenocarcinoma (mucinous, colloid, linitis plastica,leather-bottle stomach) cell. In some embodiments, the stomach orgastric carcinoma cell is a lymphoma cell.

In some embodiments, the cancer cell is an esophageal carcinoma cell.Esophageal carcinoma cells may refer to any carcinoma cell originatingfrom the esophagus, including primary tumors or esophageal carcinomacells that have metastasized to other sites. In some embodiments, theesophageal carcinoma cell is an adenocarcinoma cell. In someembodiments, the esophageal carcinoma cell is a squamous carcinoma cell.

In some embodiments, the cancer cell is a fibrosarcoma cell.Fibrosarcoma cells may refer to any carcinoma cell originating from thefibrous connective tissue, including primary tumors or esophagealcarcinoma cells that have metastasized to other sites.

In some embodiments, the cancer cell is a leukemia cell. Leukemia cellsmay refer to any malignant white blood cell, typically originating inthe bone marrow, and includes chronic, acute, lymphocytic, andmyelogenous leukemia cells. For example, in some embodiments, theleukemia cell may be an acute T leukemia cell.

In some embodiments, the cancer cell is an adenoid carcinoma cell,including primary tumors or esophageal carcinoma cells that havemetastasized to other sites. In some embodiments, the adenoid carcinomacell is an adenoid cystic carcinoma cell.

VI. Gastrointestinal Disease and Rheumatoid Arthritis

Certain aspects of the present disclosure relate to methods for treatingor preventing gastrointestinal disease by administering to theindividual an effective amount of a composition containing an antibodythat specifically binds to an epitope containing N-acetylglucosamine orN-acetyl-galactosamine, where the epitope is expressed by aninflammatory cell. In some embodiments, the individual has, or has beendiagnosed with, a gastrointestinal disease of the present disclosure.

In some embodiments, the epitope is expressed on a cell surface of aninflammatory cell. In some embodiments, the inflammatory cell is anintestinal inflammatory cell of colitis, inflammatory bowel disease, orgastroenteritis, and the epitope is expressed on a cell surface of theinflammatory cell. As described herein, antibodies that specificallyrecognize an epitope containing N-acetylglucosamine orN-acetyl-galactosamine may bind to inflammatory cells (e.g., leukocytes,such as neutrophils, macrophages, monocytes, eosinophils, and/orbasophils) at sites of inflammation in the colon, such as those seen indiseases characterized by inflammation of the colon (e.g., colitis, IBD,or gastroenteritis).

Other aspects of the present disclosure relate to methods for treatingor preventing rheumatoid arthritis by administering to the individual aneffective amount of a composition containing an antibody thatspecifically binds to an epitope containing N-acetylglucosamine orN-acetyl-galactosamine. In some embodiments, the individual has, or hasbeen diagnosed with, rheumatoid arthritis. In some embodiments, theepitope is expressed by an inflammatory cell. The results describedherein suggest that antibodies against N-acetylglucosamine and/orN-acetyl-galactosamine (e.g., antibody 105C9, or variants thereof suchas VK1, VK2, VK3, and/or VK4) may be effective in treating one or moresymptoms of rheumatoid arthritis. In some embodiments, the inflammatorycell is a T- or B-cell.

In some embodiments, the inflammatory cell is an inflammatory cell ofrheumatoid arthritis. As described herein, antibodies that specificallyrecognize an epitope containing N-acetylglucosamine orN-acetyl-galactosamine may be useful in treating or preventing one ormore symptoms of rheumatoid arthritis. For example, these antibodieshave demonstrated efficacy in a collagen-induced arthritis model, whichis known in the art as a commonly studied autoimmune model of rheumatoidarthritis (see, e.g., Brand, D. D. et al. (2007) Nat. Protoc.2:1269-1275).

VII. Methods of Treatment

Cancer

Certain aspects of the present disclosure relate to methods for treatingor preventing cancer in an individual by administering to the individualan effective amount of a composition containing an antibody thatspecifically binds to an epitope containing N-acetylglucosamine orN-acetyl-galactosamine. It is a surprising finding described herein thatantibodies that specifically bind to an epitope containingN-acetylglucosamine or N-acetyl-galactosamine, expressed on a cellsurface of or in the cancer cell, may be used to inhibit the growth of avariety of cancer cells. In some embodiments, the binding of theantibody to the epitope expressed on the cell surface of the cancer cellinhibits growth of the cancer cell.

In some embodiments, the cancer may include pancreatic cancer, coloncancer, rectal cancer, esophageal cancer, leukemia, adenocarcinoma,fibrosarcoma, duodenal adenocarcinoma, brain cancer, liver cancer, lungcancer, breast cancer, ovarian cancer, cervical cancer, colon cancer,stomach cancer, esophageal cancer, and fibrosarcoma. In someembodiments, the individual has, or has been diagnosed with, a cancer ofthe present disclosure. Since the present disclosure demonstrates thatmany types of cancer tissues express high levels of N-acetylglucosamineor N-acetyl-galactosamine, the methods described herein may be broadlyeffective in treating many types of cancer. In some embodiments, thecancer to be treated or prevented refers to a primary tumor, e.g., aprimary tumor representing pancreatic cancer, colon cancer, rectalcancer, esophageal cancer, leukemia, adenocarcinoma, fibrosarcoma,duodenal adenocarcinoma, brain cancer, liver cancer, lung cancer, breastcancer, ovarian cancer, cervical cancer, colon cancer, stomach cancer,esophageal cancer, or fibrosarcoma. In some embodiments, the cancer tobe treated or prevented refers to a metastatic cancer originallyrepresenting pancreatic cancer, colon cancer, rectal cancer, esophagealcancer, leukemia, adenocarcinoma, fibrosarcoma, duodenal adenocarcinoma,brain cancer, liver cancer, lung cancer, breast cancer, ovarian cancer,cervical cancer colon cancer, stomach cancer, esophageal cancer, orfibrosarcoma.

In some embodiments, the cancer to be treated or prevented is a braincancer. Brain cancer may refer to any cancer originating from the brain,including but not limited to a cancer made of the cells described above.Examples of brain cancers may include without limitation gliomas,meningiomas, nerve sheath tumors, and pituitary adenomas. Brain cancermay also refer to a cancer originating from the central nervous system,e.g., the spine.

In some embodiments, the cancer to be treated or prevented is a livercancer. Liver cancer may refer to any cancer originating from the liver,including but not limited to a cancer made of the cells described above.Examples of liver cancers may include without limitationhepatocarcinomas, cholangiocarcinomas, and hepatoblastomas.

In some embodiments, the cancer to be treated or prevented is a lungcancer. Lung cancer may refer to any cancer originating from the lung,including but not limited to a cancer made of the cells described above.Examples of lung cancers may include without limitation non-small-celllung cancers, including adenocarcinomas, squamous-cell carcinomas, andlarge-cell carcinomas, as well as small-cell lung carcinomas.

In some embodiments, the cancer to be treated or prevented is a breastcancer. Breast cancer may refer to any cancer originating from thebreast, including but not limited to a cancer made of the cellsdescribed above. Examples of breast cancers may include withoutlimitation ductal carcinomas in situ, invasive ductal carcinomas, triplenegative breast cancer (e.g., cancer made of cells negative forprogesterone, estrogen, and HER2/neu receptors), and inflammatory breastcancer.

In some embodiments, the cancer to be treated or prevented is an ovariancancer. Ovarian cancer may refer to any cancer originating from theovary, including but not limited to a cancer made of the cells describedabove. Examples of ovarian cancers may include without limitationsurface epithelial-stromal tumors (including, e.g., endometrioid tumors,mucinous cystadenocarcinomas, and serous tumors), germ cell tumors, sexcord-stromal tumors, and mixed ovarian tumors.

In some embodiments, the cancer to be treated or prevented is a cervicalcancer. Cervical cancer may refer to any cancer originating from thecervix, including but not limited to a cancer made of the cellsdescribed above. Examples of cervical cancers may include withoutlimitation squamous cell carcinomas, adenocarcinomas, small cellcarcinomas, adenosquamous carcinomas, neuroendocrine tumors,villoglandular adenocarcinomas, and glassy cell carcinomas.

In some embodiments, the cancer to be treated or prevented is a coloncancer. Colon cancer may refer to any cancer originating from the colonor rectum, including but not limited to a cancer made of the cellsdescribed above. Examples of colon cancers may include withoutlimitation adenocarcinomas and adenosquamous carcinomas.

In some embodiments, the cancer to be treated or prevented is a stomachor gastric cancer. Stomach or gastric cancer may refer to any canceroriginating from the stomach, including but not limited to a cancer madeof the cells described above. Examples of stomach or gastric cancers mayinclude without limitation adenocarcinomas, diffuse type adenocarcinomas(mucinous, colloid, linitis plastica, leather-bottle stomach) andlymphoma.

In some embodiments, the cancer to be treated or prevented is anesophageal cancer. Esophageal cancer may refer to any cancer originatingfrom the esophagus, including but not limited to a cancer made of thecells described above. Examples of esophageal cancers may includewithout limitation adenocarcinomas and squamous carcinomas.

In some embodiments, the cancer to be treated or prevented is afibrosarcoma. Fibrosarcomas may refer to any carcinomas originating fromthe fibrous connective tissue, including but not limited to a cancermade of the cells described above.

In some embodiments, the cancer to be treated or prevented is apancreatic cancer. Pancreatic cancer may refer to any cancer originatingfrom the pancreas, including but not limited to a cancer made of thecells described above, such as a pancreatic ductal carcinoma cell.

In some embodiments, the cancer to be treated or prevented is aleukemia. Leukemia may refer to any cancer characterized by malignantwhite blood cells, including but not limited to a cancer made of thecells described above, such as an acute T leukemia cell.

In some embodiments, the cancer to be treated or prevented is anadenocarcinoma. Adenocarcinoma may refer to any carcinoma originatingfrom a glandular structure in an epithelium, including but not limitedto a cancer made of the cells described above, such as an adenoid cysticcarcinoma cell or a duodenal adenocarcinoma cell.

Administration and Combination Therapies

Any method known in the art may be used to administer an effectiveamount of a composition containing an antibody that specifically bindsto an epitope containing N-acetylglucosamine or N-acetyl-galactosamine,such as intravenous administration as a bolus or by continuous infusionover a period of time, by intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes. In some embodiments,the antibody is administered orally. In some embodiments, thecomposition contains an antibody that specifically binds to an epitopecontaining N-acetylglucosamine or N-acetyl-galactosamine and anotherprotein, e.g., another antibody that does not specifically bind to anepitope containing N-acetylglucosamine or N-acetyl-galactosamine. Aneffective amount of a composition containing an antibody thatspecifically binds to an epitope containing N-acetylglucosamine orN-acetyl-galactosamine may be determined by any method known in the artand may depend upon a number of characteristics of the individual asdescribed above.

Certain aspects of the present disclosure relate to methods for treatingor preventing cancer in an individual by administering to the individualan effective amount of a composition containing an antibody thatspecifically binds to an epitope containing N-acetylglucosamine orN-acetyl-galactosamine and an amount of another anti-cancer agent, wherethe antibody and the anti-cancer agent in conjunction provide effectivetreatment or prevention of cancer in the individual. Any suitableanti-cancer agent known in the art may be used in combination with theantibodies described herein. Anti-cancer agents may include antibodies(including antibody-drug conjugates), small molecules,immunotherapeutics, differentiating agents, targeted therapies, andhormones.

In some embodiments, the anti-cancer agent is a chemotherapeutic agent.Many types of chemotherapeutic agents are known in the art. Examples ofchemotherapeutic agents may include without limitation antimetabolites(e.g., 5-fluorouracil or capecitabine), anthracyclines, anti-tumorantibiotics (e.g., actinomycin-D, mitomycin-C, or bleomycin), mitoticinhibitors (e.g., taxanes such as Taxol® or epothilones),corticosteroids, topoisomerase inhibitors (e.g., etoposide), alkylatingagents, and platinum drugs (e.g., cisplatin, oxalaplatin, orcarboplatin). These drugs are provided as examples for one of skill inthe art and are in no way intended to limit the choice ofchemotherapeutic agents.

Gastrointestinal Disease and Rheumatoid Arthritis

Certain aspects of the present disclosure relate to methods for treatingor preventing gastrointestinal disease in an individual by administeringto the individual an effective amount of a composition containing anantibody that specifically binds to an epitope containingN-acetylglucosamine or N-acetyl-galactosamine. It is a surprisingfinding described herein that antibodies that specifically bind to anepitope containing N-acetylglucosamine or N-acetyl-galactosamine,expressed on a cell surface of an inflammatory cell, may be used totreat or prevent a wide range of gastrointestinal diseases, includingautoimmune and infectious diseases.

As described herein, the methods of the present disclosure are effectiveagainst a wide range of gastrointestinal or autoimmune diseases in anindividual. In some embodiments, the individual has inflammatory boweldisease. An inflammatory bowel disease of the present disclosure may bechronic or acute. As is known in the art, many gastrointestinal diseasessuch as inflammatory bowel disease may present symptoms in tissuesincluding without limitation the small and large intestines, mouth,stomach, esophagus, and anus. In some embodiments, an inflammatory boweldisease may include colitis (such as diversion, lymphocytic,collagenous, or indeterminate colitis) or Behcet's disease.

In some embodiments, the individual has Crohn's disease. In someembodiments, the individual has ulcerative colitis. In some embodiments,the individual has acute infectious gastroenteritis. In someembodiments, the individual has a hemorrhoid. In some embodiments, theindividual has rheumatoid arthritis.

In some embodiments, the individual has a gastrointestinal diseasecaused by a viral infection. Viruses known to cause gastrointestinaldisease may include without limitation rotaviruses, noroviruses,adenoviruses, and astroviruses. In some embodiments, the viral infectionis a rotaviral infection.

In some embodiments, the individual with a gastrointestinal orautoimmune disease is a human. In some embodiments, the individual witha gastrointestinal or autoimmune disease is a non-human animal.

Many suitable methods for administering a composition for treating orpreventing a gastrointestinal or autoimmune disease are known in theart. In some embodiments, an antibody of the present disclosure may beadministered intravenously, intramuscularly, subcutaneously, topically,orally, transdermally, intraperitoneally, intraorbitally, byimplantation, by inhalation, intrathecally, intraventricularly, orintranasally.

Chimeric Antigen Receptor (CAR) T-Cell Therapy

Further exemplified herein (see, e.g., Example 5 below) is the use ofany of the antibodies of the present disclosure in a CAR, e.g., for aCAR T-cell therapy. CAR T-cell therapy is known in the art as atherapeutic approach that utilizes the antigen-binding region of anantibody (e.g., a single chain variable fragment of scFv) fused totransmembrane and intracellular signal domains of a T-cell signalingmolecule. These CAR T-cells typically recognize antigen(s), e.g.,unprocessed antigens, based on the specificity of the antibodies fromwhich they are derived, thereby redirecting the T-cells for atherapeutic purpose, such as targeting tumor cells bearing a particularantigen, or redirecting Treg cells to block an inflammatory disease. Forgreater description, see, e.g., Dai, H. et al. (2016) J. Natl. CancerInst. 108(7):djv439.

Immunotherapy is a therapy that harnesses the power of a patient'simmune system to combat their disease. One approach to immunotherapyinvolves engineering patients' own immune cells to recognize and attacktheir tumors. Although this approach, called adoptive cell transfer(ACT), has been restricted to small clinical trials so far, treatmentsusing these engineered immune cells have generated some remarkableresponses in patients with advanced cancer.

ACT's building blocks are T cells, a type of immune cell collected fromthe patient's own blood. After collection, the T cells are geneticallyengineered to produce special receptors on their surface called chimericantigen receptors (CARs). CARs are proteins that allow the T cells torecognize a specific protein (antigen) on tumor cells. These engineeredCAR-T cells are then grown in the laboratory, e.g., until they number inthe billions. The expanded population of CAR-T cells is then infusedinto the patient. After the infusion, the T cells multiply in thepatient's body and, with guidance from their engineered receptor,recognize and kill cancer cells that harbor the antigen on theirsurfaces.

N-acetylglucosamine and N-acetyl-galactosamine or glycoconjugatesbearing distinct N-Acetyl glucosamine or N-acetyl-galactosamine moietiescan be the harbor antigens on tumor cells and the antibodies recognizethose antigens disclosed in the current and previous patents can bereceptors for the tumor antigens. Thus one aspect of the currentdisclosure is to genetically engineering the genes encoding antibody K3,K4 and other antibodies recognizing N-acetylglucosamine andN-acetyl-galactosamine or glycoconjugates bearing distinct N-Acetylglucosamine or N-acetyl-galactosamine moieties (e.g., an antibodyfragment bearing antigen-binding fragments or heavy and/or light chainvariable domains of such antibodies) into T cells from patients toproduce CARs on the T cells. These engineered CAR-T cells can be usedfor the treatment of cancers and autoimmune diseases such as IBD andrheumatoid arthritis as described herein.

In some embodiments, a CAR comprises an antibody fragment comprising alight chain variable region comprising an HVR-L1 sequence of SEQ ID NO:5, an HVR-L2 sequence of SEQ ID NO: 7, and an HVR-L3 sequence of SEQ IDNO: 9 and/or a heavy chain variable region comprising an HVR-H1 sequenceof SEQ ID NO: 1, an HVR-H2 sequence of SEQ ID NO: 2, and an HVR-H3sequence of SEQ ID NO: 3. In some embodiments, a CAR comprises anantibody fragment comprising a light chain variable region comprising anHVR-L1 sequence of SEQ ID NO: 5, an HVR-L2 sequence of SEQ ID NO: 8, andan HVR-L3 sequence of SEQ ID NO: 9 and/or a heavy chain variable regioncomprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3.

Certain aspects of the present disclosure relate to chimeric antigenreceptors (CARs). In some embodiments, the CAR specifically binds to anepitope comprising N-acetylglucosamine or N-acetyl-galactosamine. Insome embodiments, the CAR comprises a light chain variable regioncomprising an HVR-L1 sequence of SEQ ID NO: 5, an HVR-L2 sequence ofFTSX₁LX₂S (SEQ ID NO: 25), and an HVR-L3 sequence of SEQ ID NO: 9,wherein X₁ is T or S and X₂ is Q or E and/or a heavy chain variableregion comprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequenceof SEQ ID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3. In someembodiments, the HVR-L2 sequence comprises the amino acid sequence ofSEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the CAR specificallybinds to an epitope comprising N-acetylglucosamine and an epitopecomprising N-acetyl-galactosamine.

In some embodiments, the CAR comprises a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO:26. Insome embodiments, the CAR comprises a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 13.

In some embodiments, the CAR comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 33.

In some embodiments, the light chain variable region and/or the heavychain variable region are humanized or human.

As is known in the art, CARs typically include one or more polypeptidedomains that potentiate T-cell receptor signaling, such as the CD3 zetaand/or CD28 endodomains (see, e.g., Maher, J. et al. (2002) Nat.Biotechnol. 20:70-75 and Savoldo, B. et al. (2011) J. Clin. Invest.121:1822-1826). In some embodiments, the CAR comprises a human CD3 zeta(also known as CD247) endodomain sequence. In some embodiments, humanCD3 zeta refers to a polypeptide encoded by the human CD247 gene, e.g.,as described by NCBI RefSeq Gene ID No. 919 and/or UniProt Accession No.P20963. In some embodiments, the CAR further comprises a human CD28endodomain sequence. In some embodiments, human CD28 refers to apolypeptide encoded by the human CD28 gene, e.g., as described by NCBIRefSeq Gene ID No. 940 and/or UniProt Accession No. P10747.

In some embodiments, the CAR comprises, from N-terminus to C-terminus,the heavy chain variable region, a linker, the light chain variableregion, a CD8 hinge region, a human CD28 endodomain, and a human CD3zeta endodomain. For example, in certain embodiments, the CAR comprisesthe amino acid sequence of SEQ ID NO: 34.

As demonstrated herein, a CAR of the present disclosure, when expressedby a T cell (e.g., on the cell surface) is able to induce tumor cellkilling. For example, the CAR can bind an epitope (e.g., comprisingN-acetylglucosamine and/or N-acetyl-galactosamine) expressed on a cellsurface of a cancer cell (e.g., a cancer cell of the presentdisclosure). In some embodiments, binding of the CAR to the epitopeexpressed on the cell surface of the cancer cell leads to killing of thecancer cell by the T cell.

Further provided herein are isolated polynucleotides comprising anucleic acid sequence encoding a CAR of the present disclosure. Alsoprovided are vectors comprising a nucleic acid sequence encoding a CARof the present disclosure. In some embodiments, the vector is a viralvector. Various types of viral vectors are known in the art and includewithout limitation lentiviral, adenoviral, adeno-associated viral (AAV),herpes simplex viral (HSV) vectors. Yet further provided are isolated Tcells (e.g., human T cells) comprising a nucleic acid sequence encodinga CAR of the present disclosure and/or expressing a CAR of the presentdisclosure, e.g., on their cell surface.

The CARs of the present disclosure (as well as the isolatedpolynucleotides, vectors, and isolated T cells related thereto) may finduse in a method of treatment of the present disclosure, such as themethods of treating or preventing cancer described herein. As such,provided herein are methods for treating or preventing cancer in anindividual, comprising administering to the individual an effectiveamount of a composition comprising a T cell of the present disclosure,e.g., a T cell that expresses a CAR of the present disclosure. In someembodiments, the cancer is a type of cancer described herein, includingwithout limitation a cancer selected from the group consisting ofpancreatic cancer, colorectal cancer, esophageal cancer, leukemia,adenocarcinoma, fibrosarcoma, duodenal adenocarcinoma, brain cancer,liver cancer, lung cancer, breast cancer, ovarian cancer, and cervicalcancer. In certain embodiments, the cancer is pancreatic cancer,colorectal cancer, lung cancer, or brain cancer, including the subtypesthereof described herein. In some embodiments, the individual is ahuman.

In some embodiments, the methods further include administering anotheranti-cancer agent, such as those described in reference to combinationtherapies herein. In some embodiments, the T cell and the anti-canceragent in conjunction provide effective treatment or prevention of cancerin the individual. In some embodiments, the anti-cancer agent is achemotherapeutic agent.

VIII. Methods of Detection

Certain aspects of the present disclosure relate to methods fordetecting the presence of cancer cells in an individual by: obtaining abiological sample from an individual, contacting the biological samplewith an antibody that specifically binds to an epitope containingN-acetylglucosamine or N-acetyl-galactosamine, and detecting the amountof antibody binding to the biological sample, where antibody bindingindicates the presence of cancer cells in the individual.Advantageously, the present disclosure describes how an antibody thatspecifically binds to an epitope containing N-acetylglucosamine orN-acetyl-galactosamine may be used to detect the presence of a cancer inan individual, due to the correlation between elevated levels ofN-acetylglucosamine or N-acetyl-galactosamine in a serum sample and thepresence of a cancer. These methods may be used to detect many types ofcancer, including without limitation lung cancer, liver cancer, breastcancer, colon or colorectal cancer, esophageal cancer, stomach cancer,endometrial cancer, cervical cancer, thyroid cancer, brain cancer, andlymphoma.

Specific binding between the antibody and an epitope containingN-acetylglucosamine or N-acetyl-galactosamine may be detected by anymethod known in the art. Methods for detecting binding between anantibody and an epitope are well known in the art and may include anELISA (enzyme-linked immunosorbent assay) immunohistochemistry (IHC)assays, immunofluorescence assays. flow cytometry, CTC (Circulatingtumor cells) assays, and immuno-colloidal gold assays. These exemplaryassays are well known to one of skill in the art; for more detaileddescriptions, see, e.g., Harlow and Lane, eds. (1988) Antibodies, ALaboratory Manual. Examples of ELISAs may include direct, indirect,competitive, and sandwich ELISAs. Any surface may be used, includingwithout limitation a plate (e.g., a 96-well plate) or a column.

In some embodiments, the amount of antibody binding to an epitopecontaining N-acetylglucosamine or N-acetyl-galactosamine detected asdescribed above may be compared with an amount of antibody bindingdetected using a control sample. An increased amount of antibody bindingto the biological sample may indicate the presence of cancer cells inthe individual, as compared to the amount of antibody binding to thecontrol sample. A control sample may be processed as described abovewith the biological sample from the individual to be tested. Examples ofcontrol samples may include a sample from a cancer-free individual, or asample with a known quantity of N-acetylglucosamine and/orN-acetyl-galactosamine. As a non-limiting example, serum from a testindividual may be tested using the methods described herein and comparedwith serum from a healthy (i.e., cancer-free) individual. In thisscenario, increased antibody binding in the serum from the testindividual, compared to the healthy individual or the serum with a knownquantity of N-acetylglucosamine and/or N-acetyl-galactosamine, mayindicate the presence of cancer cells in the test individual.

Any body fluid or section may be used as a biological sample of thepresent disclosure. Examples of biological samples may include withoutlimitation blood, serum, urine, feces, milk, semen, saliva, chest fluid,abdominal fluid, cerebrospinal fluid, sputum, and any other body fluidor secretion.

As shown herein, many types of cancer cells may be detected in anindividual using the methods described herein. Examples of cancer cellsthat may be detected in an individual include without limitation lungcancer cells, liver cancer cells, breast cancer cells, colon orcolorectal cancer cells, esophageal cancer cells, stomach cancer cells,endometrial cancer cells, cervical cancer cells, thyroid cancer cells,brain cancer cells, and lymphoma cells.

In addition, saccharide-related biomarkers themselves may be used in thedetection of auto-antibodies in subjects with cancers or other diseases.These auto-antibodies may bind to the saccharides that aredifferentially expressed on or in those cancerous or otherwise diseasedtissues or cells, or released to blood, urine, feces, milk, semen,saliva, and body fluid or secretions. Body fluid or secretions mayinclude but not limited to chest fluid, abdomen fluid, cerebrospinalfluid, sputum, and organ smears. In some embodiments, saccharide-relatedbiomarkers may include without limitation N-Acetyl-Glucosamine,N-Acetyl-Galactosamine or fucose, or glycoconjugates bearing distinctN-Acetyl glucosamine, N-Acetyl-Galactosamine or fucose.

IX. Kits

Certain aspects of the present disclosure relate to kits containing apharmaceutical composition containing an antibody that specificallybinds to an epitope containing N-acetylglucosamine orN-acetyl-galactosamine. In some embodiments, the kits may furtherinclude instructions for administering an effective amount of thepharmaceutical composition to an individual for treating cancer. Theseinstructions may refer to instructions customarily included incommercial packages of medicaments that contain information about theindications customarily included in commercial packages of medicamentsthat contain information about the indications, usage, dosage,administration, contraindications, other medicaments to be combined withthe packaged product, and/or warnings concerning the use of suchmedicaments, etc.

Suitable containers for a kit of the present disclosure include, forexample, bottles, vials (e.g., dual chamber vials), syringes (such assingle or dual chamber syringes) and test tubes. The article ofmanufacture may further comprise a label or a package insert, which ison or associated with the container, may indicate directions forreconstitution and/or use of the formulation. The label or packageinsert may further indicate that the formulation is useful or intendedfor oral or other modes of administration for treating or preventingcancer in an individual. The article of manufacture may further includeother materials desirable from a commercial, therapeutic, and userstandpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use.

In some embodiments, the kits containing a pharmaceutical compositioncontaining an antibody that specifically binds to an epitope containingN-acetylglucosamine or N-acetyl-galactosamine may further containinstructions for detecting the presence of cancer cells in anindividual. These instructions may refer to instructions customarilyincluded in commercial packages of ELISA assay kits,immunohistochemistry (IHC) assay kits, immunofluorescence assay kits,flow cytometry assay kits, CTC (Circulating tumor cells) assay kits, andimmuno-colloidal gold assay kits. A kit of the present disclosure mayalso contain any other reagents useful for detecting the presence ofcancer cells in an individual, such as 96-well microtiter plates, anon-specific protein such as bovine serum albumin, a secondary antibodythat binds to an antibody of the present disclosure without affectingits antigen-binding, and reagents for detection, such as a fluorescentor luminescent label, or an enzyme and substrate that produce adetectable signal (e.g., horseradish peroxidase and TMB).

Certain aspects of the present disclosure relate to kits containing apharmaceutical composition containing N-acetylglucosamine orN-acetyl-galactosamine and instructions or other reagents for using thepharmaceutical composition for detecting the presence of anauto-antibody in an individual with cancer or inflammation. Theseinstructions may refer to instructions customarily included incommercial packages of ELISA assay kits, immunohistochemistry (IHC)assay kits, immunofluorescence assay kits, flow cytometry assay kits,CTC (Circulating tumor cells) assay kits, and immuno-colloidal goldassay kits. A kit of the present disclosure may also contain any otherreagents useful for detecting the presence of cancer cells in anindividual, such as 96-well microtiter plates, a non-specific proteinsuch as bovine serum albumin, a secondary antibody that binds to anantibody of the present disclosure without affecting itsantigen-binding, and reagents for detection, such as a fluorescent orluminescent label, or an enzyme and substrate that produce a detectablesignal (e.g., horseradish peroxidase and TMB).

The specification is considered to be sufficient to enable one skilledin the art to practice the invention. Various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and scope of the appended claims.

Example 1: Binding of Humanized Antibodies to N-Acetyl-Glucosamine andN-Acetyl-Galactosamine

The identification of biomarkers that are preferentially expressed bycancer cells, rather than normal human cells, may allow for the designof new assays and therapeutic approaches to aid in the diagnosis,treatment, and/or prevention of cancer. To meet this demand, describedherein are monoclonal antibodies that bind to N-acetylglucosamine andN-acetyl-galactosamine.

Mab-1C5C9 was previously demonstrated to be efficacious in inhibitingcancer cell growth, detecting circulating cancer cells, and treating IBD(see PCT/CN2015/087717). MAb-1C5C9 was subsequently humanized. Variants(1C5-VK1 and 1C5-VK2) of humanized 105C9 with a common heavy chainvariable region (VH) and different light chain variable regions (VK)were characterized. Humanized 105C9 antibodies were tested for efficacy,binding, and toxicity in multiple in vivo animal models of IBD asdescribed in PCT/CN2015/087717.

Provided herein are new variants of humanized 105C9, 1C5-VK3 (“VK3”) and1C5-VK4 (“VK4”). These antibodies were tested for binding and efficacyin multiple in vivo animal models of IBD, arthritis and cancers asdescribed in following Examples.

Materials and Methods

Characterization of Binding

100 μL of N-acetyl-glucosamine (NAcGlu or NAG) andN-acetyl-galactosamine (NAcGal) were separately coated onto the wells ofa 96-well plate at the concentration of 1 μg/mL overnight, then washedand blocked. Humanized antibody clones 1C5-VK3 (K3) and 1C5-VK4 (K4)were tested.

After adding the test antibodies to the plate, the plate was washed, andbinding was detected by HRP-conjugated anti-human IgG as a secondaryreagent. HRP was detected by standard chromogenic assay, and binding wasassayed by OD_(450nm). In another test, each of the antibodies waspre-mixed with 20 μL of each NAcGlu and NAcGal at 1 μg/mL for 15 minutesbefore being added to a plate coated with NAcGlu. The rest of the testwas as same as described above.

Mouse IBD Model

Acute colitis was induced in BALB/c female mice, aged 6-8 weeks andweighing 17-19 grams, by anal administration of a 0.15 mL dose of 120mg/mL trinitrobenzenesulphonic acid (TNBS; Sigma-Aldrich) in 50%ethanol. Mice were divided into 5 treatment groups as follows:

-   -   G1: untreated (n=5);    -   G2: treated with 200 μg antibody Adalimumab (Humira) (Vetter        Pharma-Fertigung GmbH & Co. KG) per animal, intraperitoneally        (IP) (n=5);    -   G3: treated with 5 μg antibody K3 per animal, intraperitoneally        (IP) (n=5); and    -   G4: treated with 5 μg antibody K4 per animal, intraperitoneally        (IP) (n=5).

The treatment of G2-G4 mice was dosed once per day for 4 days from 48hours after induction of IBD by TNBS.

Body weight and clinical signs of mice in each group were recorded eachday of the IBD model. Two mice from the control group (G1) weresacrificed at 48 hours after the induction of the IBD model by TNBS, andwhole colon tissues were collected. One day (24 hrs) after the lastdosing, the procedure was ended for all animals (day 7 of the course).Whole colon tissue was collected for gross pathology (colon weight,ulceration) and histology evaluation (standard HE staining of colontissue sections) according to the key shown in Table 1 below, andrepresentative images were taken. Colon tissue pathology was evaluatedusing standard histological methods as shown in FIG. 4.

TABLE 1 Scoring legend for macroscopic colon tissue assessment. ScorePhenomenon 0 No damage 1 Hyperemia without ulcers 2 Hyperemia andthickening of bowel wall without ulcers 3 One site of ulceration withbowel wall thickness 4 Two or more sites of ulceration and inflammation5 0.5 cm inflammation and major damage 6-10 1 cm major damage. The scoreis increased by one for every 0.5 cm damage observed, to a maximum of 10

Collagen-Induced Arthritis (CIA) Model

Lewis rats at about eight weeks old were immunized once per week forthree weeks using bovine type II collagen as an immunogen. The rats withsignificant inflammatory swelling joints were randomly divided intothree groups:

-   -   Group 1 (G1): control, not treated, n=5;    -   Group 2 (G2): treated with antibody 1C5-VK2 (“K2”) at 250 μg/kg        once every other day, intravenously, n=5; and    -   Group 3 (G3): treated with antibody K3 at 250 μg/each once every        other day, intravenously, n=5.

The paw volume and body weight were measured twice a week; andrepresentative images were taken once per week. One day (24 hrs) afterthe last dosing, the procedure was ended for all animals (day 28 postdosing). Ankle and knee tissues were collected for histology evaluationincluding the status of synovia, cartilage, and bone; thymus and spleenwere collected, weighed and calculated for organ coefficient. Blood wasalso collected and serum was isolated.

Patient-Derived Xenograft (PDX) Model of Pancreatic Cancer

A Patient-Derived Xenograft (PDX) mouse model of pancreas cancer wasused for evaluation of the anti-tumor efficacy of antibody-K4. The PDXmodel was developed using Nu/Nu mice (6 weeks) and the human tumortissue from a 47 years old female patient suffering from primary poorlydifferentiated pancreas ductal adenocarcinoma without any treatment.

In Vitro Tests

A CellTiter-Glo (CTG) Luminescent Cell Viability Assay was performed.Briefly, K3 and K4 were cultured for 6 days with tumor cells from thePDX-P3 tumor tissue of the pancreas cancer model as described above;then CTG reagents was added, and the fluorescent signal was readaccording to manufacturer's instructions (Promega, Madison, USA). Thetumor cells alone were used as control. The inhibition rate of cellulargrowth was calculated by comparison between control and each test group.

In Vivo Tests

The PDX-P2 pancreas tissue was inoculated into Nu/Nu mice at 6 weeks oldfor 16 days. The mice with the tumor volume at about 200 mm³ wererandomly divided into three groups: group 1 (G1): control, not treated;group 2 (G2): treated with antibody K4 at 50 μg/each once every otherday; group 3 (G3): treated with a mouse antibody at 100 μg/each onceevery other day. The tumor size (volume) and mouse body weight weremeasured twice a week. The tumors were collected at day 37 postadministration and measured. The TGI was calculated by the formula

TGI %=[1−(Ti−T0)/(Vi−V0)]×100

-   -   Wherein T represents tumor size of test group; V represents        tumor size of control group; zero means tumor size before        administration; “i” represents a time point.

Patient-Derived Xenograft (PDX) Model of Colorectal Cancer

In Vitro Tests

K3 was cultured for 6 days with tumor cells of a PDX-P3 rectal tumortissue from a 67 years old male with moderately differentiated colonadenocarcinoma. Then the CTG reagents was added, and the fluorescentsignal was read according to manufacturer's instructions. The tumorcells alone were used as control. The inhibition rate of cellular growthwas calculated by comparison between control and each of test group.

In Vivo Tests

A mouse PDX model of rectal cancer was developed using NCG mice (6weeks) and the human tumor tissue from a 47 years old male patient withprimary poorly differentiated rectal adenocarcinoma without anytreatment. The PDX-P2 rectal tissue was inoculated into NCG mice at 6weeks old for 22 days. The mice with the tumor volume at about 80-100mm³ were randomly divided into three groups: group 1 (G1): control, nottreated; group 2 (G2): treated with antibody K3 at 50 μg/each once everyother day; group 3 (G3): treated with antibody K4 at 50 μg/each onceevery other day. The tumor size (volume) and mouse body weight weremeasured twice a week. The tumors were collected at day 17 postadministration and measured. The TGI was calculated by the formuladescribed above.

In Vitro Binding of Antibodies to Tumor Cells

An ELISA assay was developed for detection of the binding of monoclonalantibodies to human tumor cells. Briefly, a 96-well tissue-culture platewith human tumor cells was incubated until about 90% confluence. Thenthe medium was removed and replaced with 100 μL fresh culture mediumwithout serum, the medium was discarded, and the plate was storedwithout cover at 4° C. until the plate dried. The plate was sealed untiluse in a binding test.

For the ELISA assay, 100 μI/well antibody K3 and K4 was added induplicate to the 96-well plates coated with human tumor cells asmentioned above and incubated for one hour at room temperature. This waswashed and horseradish peroxidase (HRP)-conjugated anti-human IgG wasused as secondary reagent, and TMB (3,3′,5,5′-tetramethylbenzidine)Peroxidase EIA Substrate Kit (Bio-Rad, Hercules, Calif., USA) was usedfor the detection of the HRP-labeled antibodies. The plates were read byan ELISA reader at OD₄₅₀. Phosphate buffered saline (PBS) was used as anegative control.

Results

FIGS. 1A & 1B show that both antibodies displayed dose-dependent bindingto NAcGlu (FIG. 1A) and NAcGal (FIG. 1B). FIG. 2 shows that the bindingof K3 and K4 was inhibited by pre-treatment with NAcGlu and NAcGal.These results demonstrate that K3 and K4 specifically recognizeN-acetyl-glucosamine and N-acetyl-galactosamine. The two humanizedantibodies K3 and K4 were further characterized as described infra.

Example 2: Testing Efficacy of Humanized Antibodies toN-Acetyl-Glucosamine and N-Acetyl-Galactosamine in Mouse IBD and Rat CIAModels

Mouse and rat models of IBD are used to explore disease pathogenesis andvalidate therapeutic targets. Acute colitis was induced in mice asdescribed above, and the effect of humanized K3 and K4 administration onbody weight, clinical signs such as diarrhea, and colon condition wastested and compared to the effect of Adalimumab. The effect of antibodytreatment was assayed by macroscopic assessment of colon conditionsaccording to the key provided in Table 1 above. Colons from mice intreatment groups G1-G4 were assessed for the presence of colitis, asshown in FIG. 4.

FIG. 3 shows that treatment with K3 (G3) resulted in a significantdecrease in score as compared to untreated controls, demonstrating thatantibody K3 treatment resulted in significant mitigation of ulcerationand inflammation. It should be noted that treatment with Adalimumab (G2)did not show significant mitigation of ulceration and inflammation.

Representative gross images taken from these colon samples are shown inFIG. 4. All the untreated mice of control group (G1) showed colitis withulceration, inflammation, and tissue necrosis. In group G2, one case(#1) showed thickening of the bowel wall without ulceration andinflammation, while four cases (#2, 3, 4 and 5) showed colitis withulceration or inflammation or both. In group G3, two cases (#1 and #2)showed thickening of the bowel wall, light inflammation withoutsignificant ulceration, while 3 samples (#3, 4, and 5) showed noulceration and inflammation. In group G4, three cases (#1, 2, 4 and 5)showed thickening of the bowel wall without ulceration and lightinflammation.

These results illustrate that K3 treatment resulted in significantmitigation of ulceration and inflammation. Although K4 treatmentdemonstrated improvement the microscopic assessment score did not showstatistically significant. The results also indicated that both K3 andK4 demonstrated better efficacy than Adalimumab.

Mouse and rat models of collagen-induced arthritis (CIA) are used toexplore disease pathogenesis of rheumatoid arthritis and validatetherapeutic targets. The chief pathological features of CIA include aproliferative synovitis with infiltration of polymorphonuclear andmononuclear cells, pannus formation, cartilage degradation, erosion ofbone and fibrosis. The development of the disease is accompanied by arobust T-cell and B-cell inflammation response to type II collagen.Disease activity is determined by measuring inflammation swelling in theaffected joints (paw volume or thickness) over time.

A rat CIA model was used, as described above. As shown in FIGS. 5A & 5Band FIG. 6B, treatment with antibody VK3 significantly reduced theinflammatory severity of rat ankles and knees compared to those ofcontrols. VK3 also reduced inflammation better than antibody VK2.

Representative gross images are shown in FIGS. 6A & 6B. FIG. 6A showsrepresentative gross images taken at day 5 post dosing (after 2 dosing).All rat ankles and knees were swelling, red and hot. All rats walkedwith dragged legs. FIG. 6B shows representative gross images taken atday 28 post dosing (after 14 dosing).

These results demonstrate the efficacy of K3 in K4 in IBD and arthritismodels of inflammatory disease.

Example 3: Testing Efficacy of Humanized Antibodies toN-Acetyl-Glucosamine and N-Acetyl-Galactosamine in PDX Cancer Models

A PDX model for pancreatic cancer was used for testing K3 and K4, asdescribed above. The results of the in vitro tests are as follows. Asshown in FIGS. 7A & 7B, antibodies K3 (FIG. 7A) and K4 (FIG. 7B) wereable to inhibit cellular growth of PDX-P3 pancreatic cancer cells atvarious concentrations. Antibodies K3 and K4 (at the concentration of 20μg/ml) inhibited the growth of the pancreas tumor cells at 51.7% and71.8% respectively.

The results of the in vivo tests are as follows. As shown in FIGS.8A-8C, antibody K4 inhibited tumor growth at about 62% at the end pointof the test. K4's efficacy against tumor growth was demonstrated inassaying tumor volume over time (FIG. 8A), TGI (FIG. 8B), andmacroscopic assessment of tumors (FIG. 8C).

A PDX model for colorectal cancer was next used for testing K3 and K4,as described above. The results of the in vitro tests are as follows. Asshown in FIG. 9, antibody K3 (at the concentration of 10 μg/ml)inhibited the growth of the rectal tumor cells at 49.6%.

The results of the in vivo tests are as follows. As shown in FIGS. 10A &10B, antibodies K3 and K4 inhibited the tumor growth at about 23% and63% respectively at the end point of the test. These results demonstratethe efficacy of K3 and K4 in inhibiting tumor growth in vitro and invivo, using multiple tumor models.

Example 4: In Vitro Binding of Humanized Antibodies toN-Acetyl-Glucosamine and N-Acetyl-Galactosamine to Tumor Cell Lines

The ELISA-based assay described above was used to characterize thebinding of humanized monoclonal antibodies K3 and K4 to human tumorcells. Antibodies K3 and K4 were tested for reactivity against varioushuman tumor cell lines including NCI-H446 (small cell lung cancer),H1299 (non-small cell lung carcinoma), ECAP-1090 (esophagealadenocarcinoma), Jurkat (acute T cell leukemia), ACC-2 (adenoid cysticcarcinoma cells), HT1080 (fibrosarcoma), HUTU80 (duodenumadenocarcinoma), MCF-7 (breast carcinoma) and Hela (cervical cancer).

Both humanized monoclonal antibodies K3 and K4 were capable of bindinghuman tumor cells, as shown in FIG. 11. Without wishing to be bound totheory, these results may suggest that the tumor antigens listed in FIG.11 either include N-acetylglucosamine and/or N-acetyl-galactosamine, orinclude glycoconjugates bearing distinct N-Acetyl glucosamine orN-acetyl-galactosamine moieties. These results show that K3 and K4 arecapable of binding a wide range of human tumor cell types.

Example 5: Tumor Cell Killing by T Cells Bearing Chimeric AntigenReceptor

The heavy chain and light chain variable domains from antibody K4described above were reformatted for use in a chimeric antigen receptor(CAR) in order to test whether a CAR-T cell with this specificity wouldbe able to effect T cell-mediated killing of cancer cells, e.g., tumorcell lines expressing N-acetylglucosamine and/or N-acetyl-galactosamine.

Construction and Generation of Lentiviral Vector

A full-length CAR bearing VH (SEQ ID NO:33) and VL (SEQ ID NO:26)domains based on those from antibody K4 was synthesized and subclonedinto a Lenti-Puro vector. The construct had the following domainstructure (from N-terminus to C-terminus): signal sequence-VHdomain-linker-VL domain-CD8 hinge-CD28 endodomain-CD3 zeta endodomainand was under the control of the EF-1 alpha promoter (FIG. 12A). Inparticular, incorporation of the CD28 signaling endodomain in additionto CD3 zeta endodomain has been demonstrated to redirect and amplifyCAR-T cell responses (see, e.g., Maher, J. et al. (2002) Nat.Biotechnol. 20:70-75). The insert was confirmed by Sanger sequencing.The recombinant Lenti-CD274-CAR-puro was prepared by Qiagen plasmid maxiprep kit. The full CAR amino acid sequence is provided in SEQ ID NO:34.

To generate lentivirus, approximately 5×10⁶ cells were seeded per 15 cmculture dish, and 25 mL DMEM culture medium containing 10% FBS wasadded. Cells were maintained in a 37° C., 5% CO₂ incubator overnight.Next, PEI stock (polyscience, CAT#23966-2) and lentivirus packagingsystem (Lenti-CAR, Lenti-Mix) were warmed to room temperature. Afterthorough mixing by pipetting up and down, 2 mL PBS, 10 μg Lenti-CAR, and11 μg Lenti-Mix plasmid were added into one well of a 6-well plate. 26μL 100 μM PEI was then added and mixed immediately. The plate wasincubated at room temperature for 10 minutes.

The PEI/DNA complex was added into a 15 cm culture dish, and the dishwas gently shaken. Dish was cultured for 6-8 hours in an incubator, theculture medium was refreshed. The dish was continuously cultured for 48hours. After 48 hours, supernatant was harvested and filtered through a0.45 μm membrane. The filtrate was centrifuged at 50,000×g for 2 hoursat 4° C., then the pellet was resuspended in 1 mL PBS. Virus stock wasaliquoted and stored at −80° C.

To titrate the lentivirus, HT1080 cells were recovered from liquidnitrogen and passaged until reaching exponential growth phase. 50,000cells were inoculated into each well of a 24-well plate and supplementedwith 500 μL culture medium. Cells were maintained in a 37° C., 5% CO₂incubator overnight.

10⁻², 10⁻¹, 1, or 10 μL lentivirus was added into each well along withpolybrene at a final concentration of 6 μg/mL. Plate was incubated in a37° C., 5% CO₂ incubator for 96 hours.

Cells were washed with PBS, and genomic DNA was extracted using agenomic DNA purification kit (Lifetech, CAT#K0512). NanoDrop 2000spectrophotometer (Thermo Fisher Scientific) was used to determine DNAconcentration.

PCR Detection

To detection successful transduction, PCR reaction were generated asshown in Table 2. Primers are listed in Table 3 below. WPRE (Woodchuckhepatitis virus post-transcriptional regulatory element) and ALB(albumin) represent known sequences in the lentiviral plasmid useful forviral titration; see, e.g., Barczak, W. et al. (2015) Mol. Biotechnol.57:195-200.

TABLE 2 PCR reaction mix Reagent Volume (20 μL) 2 × PCR Mix 10 Probe Mix(WPRE probe, ALB probe) 0.4 Primer mix (WPRE F&R, ALB F&R) 0.4 PCR gradewater 4.2

TABLE 3 Primers used for diagnostic PCR. SEQ ID Primer Sequence (5′-3′)NO Fluorophore WPRE F GGCACTGACAATTCCGTGGT 27 WPRE RAGGGACGTAGCAGAAGGACG 28 WPRE ACGTCCTTTCCATGGCTGCTCGC 29 5′-FAM-BHQ1-3′Probe ALB F GCTGTCATCTCTTGTGGGCTGT 30 ALB R ACTCATGGGAGCTGCTGGTTC 31 ALBCCTGTCATGCCCACACAAATCTCTCC 32 5′-FAM-BHQ1-3′ Probe

A new 96-well plate was prepared, and 5 μL genomic DNA samples orstandard curve was added per well along with 15 μL PCR reaction mix.Plate was sealed with membrane and briefly centrifuged for 1 min. PCRreaction thermocycles were as shown in Table 4.

TABLE 4 PCR thermocycles Temp (° C.) Time (sec) Initial HotStart/denaturation 95 30 Steps 1-2 are repeated through 40 cycles Step 195 5 Step 2 60 30

Establishment of CAR-T Cells

To isolate CAR-T cells, Lymphoprep™ density gradient medium (STEMCELLTechnologies) was mixed by tube inversion, and 15 mL Lymphoprep™ wasadded into a 50 mL tube in a biosafety cabinet. Peripheral blood sampleswere diluted with 1 volume of PBS. Diluted blood sample was slowly addedonto the top of Lymphoprep™, then centrifuged at room temperature for 30min at 800×g.

After centrifugation, PBMCs were collected from the interface betweentop yellow serum and the colorless Lymphoprep™ reagent and washed withPBS once. Cell density was adjusted to 1×10⁶ cell/mL with PBS. MagneticDynabeads were added into the cell suspension at a bead:cell ratio of3:1 and incubated for 30 minutes at room temperature. Magnet was used toisolate CD3+ T cells, and the cells were washed once with PBS.

Cells/Dynabeads were resuspended with X-vivo 15 medium containing 300U/mL IL-2, 5 ng/mL IL-15 and 10 ng/mL IL-7. Cells were continuouslycultured for 48 hours.

Transduction of Primary T Cells with Lentivirus

Cell density was adjusted to 1×10⁶ cells/mL in complete culture medium.Lentivirus amount was calculated as follows: Virus volume (mL)=(MOI*cellnumber)/virus titer.

Lentivirus and 6 μg/mL polybrene were added into the culture plate andcentrifuged at 800×g for 1 hour. After centrifugation, the cells werecultured in a 37° C. 5% CO₂ incubator for 24 hours and passaged untilready for use.

Lysis of Target Cells by CAR-T Cells

Target cells were recovered from liquid nitrogen and routinely passagedtwice prior to assay. Cell density was adjusted to 5×10⁵ cells/mL and100 μL/well were inoculated into a 96-well plate. Plate was placed in a5% CO₂ 37° C. incubator overnight.

CAR-T cells were resuspended with serum-free RPMI1640 medium and addedinto the 96-well plate according the E/T ratio as indicated. Plate wasplaced back in incubator and maintained for 6 hours.

After cultivation, lysis buffer was added into the “Maxi lysis” controlwell and centrifuged at 1200×g for 5 min. 50 μL supernatant wastransferred to a new 96-well plate, and LDH substrate was added. Theplate was incubated for 15 min, and OD_(490nm) value was read using amicroplate reader. Remaining supernatant was aliquoted for ELISA assayto determine the secretion of cytokines.

Percent lysis was calculated as follows:

${{Lysis}\mspace{14mu} \%} = {\frac{\left( {{{ODeach}\mspace{14mu} {well}} - {{ODmini}\mspace{14mu} {lysis}}} \right)}{{{ODmaxi}\mspace{14mu} {lysis}} - {{ODmini}\mspace{14mu} {lysis}}} \times 100\%}$

Results

Recombinant lentiviral vector was digested with EcoRI and BamHI, andexpected fragments were demonstrated using agarose gel electrophoresis(FIG. 12B). The recombinant vector was further sequenced to verify thecorrect construct. These results demonstrated that the vector shown inFIG. 12A was successfully produced.

To demonstrate that CAR-T cells transduced with this lentiviral vectorwere able to effect cell killing of target cells, A549 lungadenocarcinoma cells were used as target cells and the CAR-T cells aseffector cells in a cell lysis assay. After co-culturing the two celltypes for 8 hours, cell supernatant was harvested for determine theamount of LDH, indicating cell killing. As shown in FIG. 13, CAR-T cellswere able to eliminate target cells efficiently, even at an E/T ratio aslow as 0.5:1. These results demonstrate that the variable domains of theK4 antibody, which as shown in the previous Examples bind toN-acetylglucosamine and/or N-acetyl-galactosamine (or to glycoconjugatesbearing distinct N-Acetyl glucosamine or N-acetyl-galactosaminemoieties), are able to effect killing of target tumor cells when used inthe context of a chimeric antigen receptor transduced into primary humanT cells.

To demonstrate that the CAR-T cell strategy is able to effect killing ofa range of tumor cells, CAR-T cells were tested in killing assaysagainst pancreatic (BxPc-3), colorectal (HCT116), and glioblastoma (U87)tumor cell lines. First, BxPc-3 pancreatic cancer cells were used astarget cells and the CAR-T cells as effector cells in a cell lysisassay. After co-culturing the two cell types for 8 hours, cellsupernatant was harvested for determine the amount of LDH, indicatingcell killing. As shown in FIG. 14, CAR-T cells were able to eliminateBxPc-3 cells efficiently, even at an E/T ratio as low as 0.5:1.

Next, HCT116 colorectal cancer cells were used as target cells and theCAR-T cells as effector cells in a cell lysis assay. After co-culturingthe two cell types for 8 hours, cell supernatant was harvested fordetermine the amount of LDH, indicating cell killing. As shown in FIG.15, CAR-T cells were able to eliminate HCT116 cells efficiently, even atan E/T ratio as low as 0.5:1.

Next, U87 glioblastoma cancer cells were used as target cells and theCAR-T cells as effector cells in a cell lysis assay. After co-culturingthe two cell types for 8 hours, cell supernatant was harvested fordetermine the amount of LDH, indicating cell killing. As shown in FIG.16, CAR-T cells were able to eliminate U87 cells efficiently, even at anE/T ratio as low as 0.5:1.

Taken together, these results indicate that human T cells bearing a CARwith the K4 antibody specificity were able to efficiently effect cellkilling of lung, pancreatic, colorectal, and brain cancer cell lines,demonstrating the wide applicability of this CAR-T cell approach formediating tumor cell killing.

SEQUENCES

All polypeptide sequences are presented N-terminal to C-terminal unlessotherwise noted. All nucleic acid sequences are presented 5′ to 3′unless otherwise noted.

Humanized parental 1C5C9 and 1C5C9-VK1/VK2/VK3/VK4 HVR-H1:(SEQ ID NO: 1) YTFPDYNIHHumanized parental 1C5C9 and 1C5C9-VK1/VK2/VK3/VK4 HVR-H2:(SEQ ID NO: 2) CIYPYNGNTAHumanized parental 1C5C9 and 1C5C9-VK1/VK2/VK3/VK4 HVR-H3:(SEQ ID NO: 3) SDLYYFGSRGFD Humanized parental 1C5C9/1C5C9-VK2 HVR-L1:(SEQ ID NO: 4) RASQDISTYLN Humanized 1C5C9-VK3/VK4 HVR-L1:(SEQ ID NO: 5) RASQDISTYLA Humanized parental 1C5C9 HVR-L2:(SEQ ID NO: 6) FTSRLHS Humanized 1C5C9-VK3 HVR-L2: (SEQ ID NO: 7)FTSTLQS Humanized 1C5C9-VK4 HVR-L2: (SEQ ID NO: 8) FTSSLESHumanized parental 1C5C9 and 1C5C9-VK1/VK2/VK3/VK4 HVR-L3:(SEQ ID NO: 9) QQGNTLPWHumanized parental and 1C5C9-VK1/VK2/VK3/VK4 Heavy Chain Variable Region:(SEQ ID NO: 10) QVQLVQSGAEVKKPGASVKVSCKASGYTFPDYNIHWVRQAPGQGLEWMGCIYPYNGNTAYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSDLYYFGSRGFDY WGQGTLVTVSSAHumanized parental and 1C5C9-VK1/VK2/VK3/VK4 Heavy Chain:(SEQ ID NO: 11) QVQLVQSGAEVKKPGASVKVSCKASGYTFPDYNIHWVRQAPGQGLEWMGCIYPYNGNTAYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSDLYYFGSRGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHumanized parental 1C5C9 Light Chain Variable Region: (SEQ ID NO: 12)DIQMTQSPSSLSASVGDRVTITCRASQDISTYLNWYQQKPGKAPKLLIYFTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGNTLPWTFGGGTKLEIKHumanized 1C5C9-VK3 Light Chain Variable Region: (SEQ ID NO: 13)DIQMTQSPSSLSASVGDRVTITCRASQDISTYLAWYQQKPGKAPKLLIYFTSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQQGNTLPWTFGGGTKLEIKHumanized 1C5C9-VK4 Light Chain Variable Region: (SEQ ID NO: 14)AIQLTQSPSSLSASVGDRVTITCRASQDISTYLAWYQQKPGKAPKLLIYFTSSLESGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKLEIKHumanized parental 1C5C9 Light Chain: (SEQ ID NO: 15)DIQMTQSPSSLSASVGDRVTITCRASQDISTYLNWYQQKPGKAPKLLIYFTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGNTLPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Humanized 1C5C9-VK3 Light Chain:(SEQ ID NO: 16)DIQMTQSPSSLSASVGDRVTITCRASQDISTYLAWYQQKPGKAPKLLIYFTSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQQGNTLPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Humanized 1C5C9-VK4 Light Chain:(SEQ ID NO: 17)AIQLTQSPSSLSASVGDRVTITCRASQDISTYLAWYQQKPGKAPKLLIYFTSSLESGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Humanized 1C5C9-VK2 HVR-L2:(SEQ ID NO: 18) FTSSLQS Humanized 1C5-VK2 Light Chain Variable Region:(SEQ ID NO: 19)DIQMTQSPSSLSASVGDRVTITCRASQDISTYLNWYQQKPGKAPKLLIYFTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGNTLPWTFGGGTKLEIKHumanized 1C5-VK2 Light Chain: (SEQ ID NO: 20)DIQMTQSPSSLSASVGDRVTITCRASQDISTYLNWYQQKPGKAPKLLIYFTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGNTLPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Humanized 1C5C9-VK1 HVR-L1:(SEQ ID NO: 21) QASQDISTYLN Humanized 1C5C9-VK1 HVR-L2: (SEQ ID NO: 22)FTSNLET Humanized 1C5-VK1 Light Chain Variable Region: (SEQ ID NO: 23)DIQMTQSPSSLSASVGDRVTITCQASQDISTYLNWYQQKPGKAPKLLIYFTSNLETGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGNTLPWTFGGGTKLEIKHumanized 1C5-VK1 Light Chain: (SEQ ID NO: 24)DIQMTQSPSSLSASVGDRVTITCQASQDISTYLNWYQQKPGKAPKLLIYFTSNLETGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGNTLPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECConsensus humanized 1C5C9-VK3/-VK4 HVR-L2: (SEQ ID NO: 25) FTSX₁LX₂SVK4 light chain variable region used for CAR (SEQ ID NO: 26)AIQLTQSPSSLSASVGDRVTITCRASQDISTYLAWYQQKPGKAPKLLIYFTSSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKLEIKRTVAAPSVFIWPRE forward primer (SEQ ID NO: 27) GGCACTGACAATTCCGTGGTWPRE reverse primer (SEQ ID NO: 28) AGGGACGTAGCAGAAGGACG WPRE probe(SEQ ID NO: 29) ACGTCCTTTCCATGGCTGCTCGC ALB forward primer(SEQ ID NO: 30) GCTGTCATCTCTTGTGGGCTGT ALB reverse primer(SEQ ID NO: 31) ACTCATGGGAGCTGCTGGTTC ALB probe (SEQ ID NO: 32)CCTGTCATGCCCACACAAATCTCTCC VK4 heavy chain variable region used for CAR(SEQ ID NO: 33) QVQLVSGAEVKKPGASVKVSCKASGYTFPDYNIHWVRQAPGQGLEWMGCIYPYNGNTAYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSDLYYFGSRGFDY WGQGTLVTVSSGCAR amino acid sequence (SEQ ID NO: 34)MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFPDYNIHWVRQAPGQGLEWMGCIYPYNGNTAYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSDLYYFGSRGFDYWGQGTLVTVSSAGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISTYLAWYQQKPGKAPKLLIYFTSSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKLEIKRTVAAPSVFITTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

What is claimed is:
 1. An isolated monoclonal antibody that specificallybinds to an epitope comprising N-acetylglucosamine orN-acetyl-galactosamine, wherein the antibody comprises a light chainvariable region comprising an HVR-L1 sequence of SEQ ID NO: 5, an HVR-L2sequence of FTSX₁LX₂S (SEQ ID NO: 25), and an HVR-L3 sequence of SEQ IDNO: 9, wherein X₁ is T or S and X₂ is Q or E.
 2. The antibody of claim1, wherein the HVR-L2 sequence comprises the amino acid sequence of SEQID NO:
 7. 3. The antibody of claim 2, wherein the antibody comprises alight chain variable region comprising the amino acid sequence of SEQ IDNO:
 13. 4. The antibody of claim 3, wherein the antibody comprises alight chain comprising the amino acid sequence of SEQ ID NO:
 16. 5. Theantibody of claim 1, wherein the HVR-L2 sequence comprises the aminoacid sequence of SEQ ID NO:
 8. 6. The antibody of claim 5, wherein theantibody comprises a light chain variable region comprising the aminoacid sequence of SEQ ID NO:
 14. 7. The antibody of claim 6, wherein theantibody comprises a light chain comprising the amino acid sequence ofSEQ ID NO:
 17. 8. The antibody of any one of claims 1-7, wherein theantibody further comprises a heavy chain variable region comprising anHVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQ ID NO: 2, andan HVR-H3 sequence of SEQ ID NO:
 3. 9. The antibody of claim 8, whereinthe antibody comprises a heavy chain variable region comprising theamino acid sequence of SEQ ID NO:
 10. 10. The antibody of claim 9,wherein the antibody comprises a heavy chain comprising the amino acidsequence of SEQ ID NO:
 11. 11. The antibody of any one of claims 1-10,wherein the antibody is an antibody fragment.
 12. The antibody of claim11, wherein the antibody is a Fab fragment, scFv, minibody, diabody,scFv multimer, or bispecific antibody fragment.
 13. The antibody of anyone of claims 1-12, wherein the antibody is a humanized antibody. 14.The antibody of any one of claims 1, 2, 5, and 11-13, wherein theantibody is a human antibody.
 15. The antibody of any one of claims 1,2, 5, and 11-13, wherein the antibody is a chimeric antibody.
 16. Theantibody of any one of claims 1-15, wherein the antibody specificallybinds to an epitope comprising N-acetylglucosamine and an epitopecomprising N-acetyl-galactosamine.
 17. The antibody of any one of claims1-16, wherein the epitope is expressed by a cancer cell or aninflammatory cell.
 18. The antibody of claim 17, wherein the epitope isexpressed on a cell surface of the cancer cell.
 19. The antibody ofclaim 17, wherein the epitope is expressed in the cancer cell.
 20. Theantibody of claim 18 or claim 19, wherein the cancer cell is selectedfrom the group consisting of a pancreatic adenocarcinoma cell, a colonadenocarcinoma cell, a rectal adenocarcinoma cell, an esophagealadenocarcinoma cell, a leukemia cell, an adenoid carcinoma cell, afibrosarcoma cell, a duodenal adenocarcinoma cell, a glioma cell, ahepatocarcinoma cell, a lung cancer cell, a breast cancer cell, aglioblastoma cell, an ovarian carcinoma cell, and a cervicaladenocarcinoma cell.
 21. The antibody of claim 20, wherein thepancreatic adenocarcinoma cell is a pancreatic ductal adenocarcinomacell.
 22. The antibody of any one of claims 18-21, wherein the bindingof the antibody to the epitope inhibits growth of the cancer cell. 23.The antibody of claim 17, wherein the inflammatory cell is an intestinalinflammatory cell of colitis, inflammatory bowel disease, orgastroenteritis, and the epitope is expressed on a cell surface of theinflammatory cell.
 24. The antibody of claim 17, wherein theinflammatory cell is an inflammatory cell of rheumatoid arthritis, andthe epitope is expressed on a cell surface of the inflammatory cell. 25.The antibody of claim 24, wherein the rheumatoid arthritis is induced bycollagen.
 26. An isolated polynucleotide comprising a nucleic acidsequence encoding the antibody of any one of claims 1-25.
 27. A vectorcomprising a nucleic acid sequence encoding the antibody of any one ofclaims 1-25.
 28. An isolated host cell comprising the polynucleotide ofclaim 25 or the vector of claim
 27. 29. A method of producing anantibody, comprising culturing the host cell of claim 28 that producesthe antibody encoded by the nucleic acid, and recovering the antibodyfrom the cell culture.
 30. An antibody produced by the method of claim29.
 31. A composition comprising the antibody of any one of claims 1-25and 30, and a pharmaceutically acceptable carrier.
 32. A method fortreating or preventing cancer in an individual, comprising administeringto the individual an effective amount of a composition comprising anantibody of any one of claims 1-25 and
 30. 33. The method of claim 32,wherein the cancer is selected from the group consisting of pancreaticcancer, colon cancer, rectal cancer, esophageal cancer, leukemia,adenocarcinoma, fibrosarcoma, duodenal adenocarcinoma, brain cancer,liver cancer, lung cancer, breast cancer, ovarian cancer, and cervicalcancer.
 34. The method of claim 32 or claim 33, wherein the individualis a human.
 35. The method of claim 32 or claim 33, wherein theindividual is a non-human animal.
 36. A method for treating orpreventing cancer in an individual, comprising administering to theindividual an amount of the antibody of any one of claims 1-25 and 30,and an amount of another anti-cancer agent, whereby the antibody and theanti-cancer agent in conjunction provide effective treatment orprevention of cancer in the individual.
 37. The method of claim 36,wherein the cancer treated is selected from the group consisting ofpancreatic cancer, colon cancer, rectal cancer, leukemia,adenocarcinoma, brain cancer, liver cancer, lung cancer, breast cancer,ovarian cancer, cervical cancer, stomach or gastric cancer, esophagealcancer, and fibrosarcoma.
 38. The method of claim 36 or claim 37,wherein the individual is a human.
 39. The method of claim 36 or claim37, wherein the individual is a non-human animal.
 40. The method of anyone of claims 36-39, wherein the anti-cancer agent is a chemotherapeuticagent.
 41. A method for detecting cancer cells in an individual,comprising: (a) contacting a biological sample from the individual withthe antibody of any one of claims 1-25 and 30; and (b) detecting bindingof the antibody to the biological sample, wherein binding of theantibody to the sample may indicate the presence of cancer cells in theindividual.
 42. The method of claim 41, further comprising comparing theamount of antibody binding detected in step (b) with an amount ofantibody binding to a control sample.
 43. The method of claim 41 orclaim 42, wherein the binding of the antibody to the biological sampleis detected by an assay selected from the group consisting of an ELISAassay, a flow cytometry assay, an immunohistochemistry assay, animmunofluorescence assay, a circulating tumor cells assay, and animmune-colloidal gold assay.
 44. The method of any one of claims 41-43,wherein the biological sample is selected from the group consisting ofblood, serum, urine, feces, milk, semen, saliva, chest fluid, abdominalfluid, cerebrospinal fluid, sputum, and any other body fluid orsecretion.
 45. The method of any one of claims 41-44, wherein theindividual is a human.
 46. The method of any one of claims 41-44,wherein the individual is a non-human animal.
 47. The method of any oneof claims 41-46, wherein the cancer cells are selected from the groupconsisting of pancreatic cancer cells, leukemia cells, adenocarcinomacells, fibrosarcoma cells, lung cancer cells, liver cancer cells, breastcancer cells, colon or colorectal cancer cells, esophageal cancer cells,stomach cancer cells, endometrial cancer cells, cervical cancer cells,thyroid cancer cells, brain cancer cells, and lymphoma cells.
 48. Amethod for treating or preventing gastrointestinal disease in anindividual comprising administering to the individual an effectiveamount of the antibody of any one of claims 1-25 and
 30. 49. The methodof claim 48, wherein the individual has inflammatory bowel disease. 50.The method of claim 48, wherein the individual has Crohn's disease. 51.The method of claim 48, wherein the individual has ulcerative colitis.52. The method of claim 48, wherein the individual has acute infectiousgastroenteritis.
 53. The method of claim 48, wherein the individual hasa hemorrhoid.
 54. The method of claim 48, wherein the individual has agastrointestinal disease caused by a viral infection.
 55. The method ofclaim 54, wherein the viral infection is a rotaviral infection or aporcine epidemic diarrhea viral infection.
 56. The method of any one ofclaims 48-55, wherein the individual is a human.
 57. The method of anyone of claims 48-55, wherein the individual is a non-human animal.
 58. Amethod for treating or preventing rheumatoid arthritis in an individualcomprising administering to the individual an effective amount of anantibody that specifically binds to an epitope comprisingN-acetylglucosamine or N-acetyl-galactosamine.
 59. The method of claim58, wherein the antibody specifically binds to an epitope comprisingN-acetylglucosamine and an epitope comprising N-acetyl-galactosamine.60. The method of claim 58 or claim 59, wherein the antibody is anantibody fragment.
 61. The method of claim 60, wherein the antibody is aFab fragment, scFv, minibody, diabody, scFv multimer, or bispecificantibody fragment.
 62. The method of any one of claims 58-61, whereinthe antibody is a humanized antibody.
 63. The method of any one ofclaims 58-61, wherein the antibody is a human antibody.
 64. The methodof any one of claims 58-61, wherein the antibody is a chimeric antibody.65. The method of any one of claims 58-64, wherein the antibodycomprises a heavy chain variable region comprising three HVRs from theamino acid sequence of SEQ ID NO:10, and a light chain variable regioncomprising three HVRs from an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 12, 13, 14, 19, and
 23. 66. The methodof any one of claims 58-64, wherein the antibody comprises a heavy chainvariable region comprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2sequence of SEQ ID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3; and alight chain variable region comprising an HVR-L1 sequence of SEQ ID NO:4, an HVR-L2 sequence of SEQ ID NO: 6, and an HVR-L3 sequence of SEQ IDNO:
 9. 67. The method of claim 66, wherein the antibody comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:10, and a light chain variable region comprising the amino acidsequence of SEQ ID NO:12.
 68. The method of claim 67, wherein theantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO:11, and a light chain comprising the amino acid sequence ofSEQ ID NO:15.
 69. The method of any one of claims 58-64, wherein theantibody comprises a heavy chain variable region comprising an HVR-H1sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQ ID NO: 2, and anHVR-H3 sequence of SEQ ID NO: 3; and a light chain variable regioncomprising an HVR-L1 sequence of SEQ ID NO: 21, an HVR-L2 sequence ofSEQ ID NO: 22, and an HVR-L3 sequence of SEQ ID NO:
 9. 70. The method ofclaim 69, wherein the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:10, and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:23. 71.The method of claim 70, wherein the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:11, and a light chaincomprising the amino acid sequence of SEQ ID NO:24.
 72. The method ofany one of claims 58-64, wherein the antibody comprises a heavy chainvariable region comprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2sequence of SEQ ID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3; and alight chain variable region comprising an HVR-L1 sequence of SEQ ID NO:4, an HVR-L2 sequence of SEQ ID NO: 18, and an HVR-L3 sequence of SEQ IDNO:
 9. 73. The method of claim 72, wherein the antibody comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:10, and a light chain variable region comprising the amino acidsequence of SEQ ID NO:19.
 74. The method of claim 73, wherein theantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO:11, and a light chain comprising the amino acid sequence ofSEQ ID NO:20.
 75. The method of any one of claims 58-64, wherein theantibody comprises a heavy chain variable region comprising an HVR-H1sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQ ID NO: 2, and anHVR-H3 sequence of SEQ ID NO: 3; and a light chain variable regioncomprising an HVR-L1 sequence of SEQ ID NO: 5, an HVR-L2 sequence of SEQID NO: 7, and an HVR-L3 sequence of SEQ ID NO:
 9. 76. The method ofclaim 75, wherein the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:10, and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:13. 77.The method of claim 76, wherein the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:11, and a light chaincomprising the amino acid sequence of SEQ ID NO:16.
 78. The method ofany one of claims 58-64, wherein the antibody comprises a heavy chainvariable region comprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2sequence of SEQ ID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3; and alight chain variable region comprising an HVR-L1 sequence of SEQ ID NO:5, an HVR-L2 sequence of SEQ ID NO: 8, and an HVR-L3 sequence of SEQ IDNO:
 9. 79. The method of claim 78, wherein the antibody comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:10, and a light chain variable region comprising the amino acidsequence of SEQ ID NO:14.
 80. The method of claim 79, wherein theantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO:11, and a light chain comprising the amino acid sequence ofSEQ ID NO:17.
 81. The method of any one of claims 58-80, wherein theindividual is a human.
 82. The method of any one of claims 58-80,wherein the individual is a non-human animal.
 83. The method of any oneof claims 32-40 and 48-82, wherein the antibody is administeredintravenously, intramuscularly, subcutaneously, topically, orally,transdermally, intraperitoneally, intraorbitally, by implantation, byinhalation, intrathecally, intraventricularly, or intranasally.
 84. Akit comprising a pharmaceutical composition comprising the antibody ofany one of claims 1-25 and
 30. 85. The kit of claim 84, furthercomprising instructions for administering an effective amount of thepharmaceutical composition to an individual for treating or preventingcancer.
 86. The kit of claim 84, further comprising instructions foradministering an effective amount of the pharmaceutical composition toan individual for treating or preventing gastrointestinal disease. 87.The kit of claim 84, further comprising instructions for detecting thepresence of cancer cells in an individual.
 88. The kit of claim 84,further comprising instructions for determining a level ofN-acetylglucosamine or N-acetyl-galactosamine in a biological samplefrom an individual with cancer.
 89. A kit comprising: (a) a compositioncomprising an antibody that specifically binds to an epitope comprisingN-acetylglucosamine or N-acetyl-galactosamine, wherein the epitope isexpressed by an inflammatory cell; and (b) instructions foradministering an effective amount of the composition to an individualfor treating or preventing rheumatoid arthritis.
 90. The kit of claim89, wherein the antibody specifically binds to an epitope comprisingN-acetylglucosamine and an epitope comprising N-acetyl-galactosamine.91. The kit of claim 89 or claim 90, wherein the antibody is an antibodyfragment.
 92. The kit of claim 91, wherein the antibody is a Fabfragment, scFv, minibody, diabody, scFv multimer, or bispecific antibodyfragment.
 93. The kit of any one of claims 89-92, wherein the antibodyis a humanized antibody.
 94. The kit of any one of claims 89-92, whereinthe antibody is a human antibody.
 95. The kit of any one of claims89-92, wherein the antibody is a chimeric antibody.
 96. The kit of anyone of claims 89-95, wherein the antibody comprises a heavy chainvariable region comprising three HVRs from the amino acid sequence ofSEQ ID NO:10, and a light chain variable region comprising three HVRsfrom an amino acid sequence selected from the group consisting of SEQ IDNOs: 12, 13, 14, 19, and
 23. 97. The kit of any one of claims 89-95,wherein the antibody comprises a heavy chain variable region comprisingan HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQ ID NO: 2,and an HVR-H3 sequence of SEQ ID NO: 3; and a light chain variableregion comprising an HVR-L1 sequence of SEQ ID NO: 4, an HVR-L2 sequenceof SEQ ID NO: 6, and an HVR-L3 sequence of SEQ ID NO:
 9. 98. The kit ofclaim 97, wherein the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:10, and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:12. 99.The kit of claim 98, wherein the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:11, and a light chaincomprising the amino acid sequence of SEQ ID NO:15.
 100. The kit of anyone of claims 89-95, wherein the antibody comprises a heavy chainvariable region comprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2sequence of SEQ ID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3; and alight chain variable region comprising an HVR-L1 sequence of SEQ ID NO:21, an HVR-L2 sequence of SEQ ID NO: 22, and an HVR-L3 sequence of SEQID NO:
 9. 101. The kit of claim 100, wherein the antibody comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:10, and a light chain variable region comprising the amino acidsequence of SEQ ID NO:23.
 102. The kit of claim 101, wherein theantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO:11, and a light chain comprising the amino acid sequence ofSEQ ID NO:24.
 103. The kit of any one of claims 89-95, wherein theantibody comprises a heavy chain variable region comprising an HVR-H1sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQ ID NO: 2, and anHVR-H3 sequence of SEQ ID NO: 3; and a light chain variable regioncomprising an HVR-L1 sequence of SEQ ID NO: 4, an HVR-L2 sequence of SEQID NO: 18, and an HVR-L3 sequence of SEQ ID NO:
 9. 104. The kit of claim103, wherein the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:10, and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:19. 105.The kit of claim 104, wherein the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:11, and a light chaincomprising the amino acid sequence of SEQ ID NO:20.
 106. The kit of anyone of claims 89-95, wherein the antibody comprises a heavy chainvariable region comprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2sequence of SEQ ID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3; and alight chain variable region comprising an HVR-L1 sequence of SEQ ID NO:5, an HVR-L2 sequence of SEQ ID NO: 7, and an HVR-L3 sequence of SEQ IDNO:
 9. 107. The kit of claim 106, wherein the antibody comprises a heavychain variable region comprising the amino acid sequence of SEQ IDNO:10, and a light chain variable region comprising the amino acidsequence of SEQ ID NO:13.
 108. The kit of claim 107, wherein theantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO:11, and a light chain comprising the amino acid sequence ofSEQ ID NO:16.
 109. The kit of any one of claims 89-95, wherein theantibody comprises a heavy chain variable region comprising an HVR-H1sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQ ID NO: 2, and anHVR-H3 sequence of SEQ ID NO: 3; and a light chain variable regioncomprising an HVR-L1 sequence of SEQ ID NO: 5, an HVR-L2 sequence of SEQID NO: 8, and an HVR-L3 sequence of SEQ ID NO:
 9. 110. The kit of claim109, wherein the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:10, and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:14. 111.The kit of claim 110, wherein the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:11, and a light chaincomprising the amino acid sequence of SEQ ID NO:17.
 112. A chimericantigen receptor (CAR) that specifically binds to an epitope comprisingN-acetylglucosamine or N-acetyl-galactosamine, wherein the CAR comprisesa light chain variable region comprising an HVR-L1 sequence of SEQ IDNO: 5, an HVR-L2 sequence of FTSX₁LX₂S (SEQ ID NO: 25), and an HVR-L3sequence of SEQ ID NO: 9, wherein X₁ is T or S and X₂ is Q or E and aheavy chain variable region comprising an HVR-H1 sequence of SEQ ID NO:1, an HVR-H2 sequence of SEQ ID NO: 2, and an HVR-H3 sequence of SEQ IDNO:
 3. 113. The CAR of claim 112, wherein the HVR-L2 sequence comprisesthe amino acid sequence of SEQ ID NO:
 8. 114. The CAR of claim 113,wherein the CAR comprises a light chain variable region comprising theamino acid sequence of SEQ ID NO: 14 or SEQ ID NO:26.
 115. The CAR ofclaim 112, wherein the HVR-L2 sequence comprises the amino acid sequenceof SEQ ID NO:
 7. 116. The CAR of claim 115, wherein the CAR comprises alight chain variable region comprising the amino acid sequence of SEQ IDNO:
 13. 117. The CAR of any one of claims 112-116, wherein the CARcomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 10 or SEQ ID NO:
 33. 118. The CAR of claim 112,wherein the light chain variable region and the heavy chain variableregion are humanized or human.
 119. The CAR of any one of claims112-118, wherein the CAR comprises a human CD3 zeta endodomain sequence.120. The CAR of any one of claims 112-119, wherein the CAR furthercomprises a human CD28 endodomain sequence.
 121. The CAR of any one ofclaims 112-120, wherein the CAR comprises, from N-terminus toC-terminus, the heavy chain variable region, a linker, the light chainvariable region, a CD8 hinge region, a human CD28 endodomain, and ahuman CD3 zeta endodomain.
 122. The CAR of claim 112, wherein the CARcomprises the amino acid sequence of SEQ ID NO:
 34. 123. The CAR of anyone of claims 112-122, wherein the CAR specifically binds to an epitopecomprising N-acetylglucosamine and an epitope comprisingN-acetyl-galactosamine.
 124. The CAR of any one of claims 112-123,wherein the epitope is expressed on a cell surface of a cancer cell.125. The CAR of claim 124, wherein the cancer cell is selected from thegroup consisting of a pancreatic adenocarcinoma cell, a colonadenocarcinoma cell, a rectal adenocarcinoma cell, an esophagealadenocarcinoma cell, a leukemia cell, an adenoid carcinoma cell, afibrosarcoma cell, a duodenal adenocarcinoma cell, a glioma cell, ahepatocarcinoma cell, a lung cancer cell, a breast cancer cell, aglioblastoma cell, an ovarian carcinoma cell, and a cervicaladenocarcinoma cell.
 126. The CAR of claim 125, wherein the pancreaticadenocarcinoma cell is a pancreatic ductal adenocarcinoma cell.
 127. TheCAR of any one of claims 124-126, wherein the CAR is expressed on a cellsurface of a T cell, and wherein binding of the CAR to the epitopeexpressed on the cell surface of the cancer cell leads to killing of thecancer cell by the T cell.
 128. An isolated polynucleotide comprising anucleic acid sequence encoding the CAR of any one of claims 112-127.129. A vector comprising a nucleic acid sequence encoding the CAR of anyone of claims 112-127.
 130. The vector of claim 129, wherein the vectoris a viral vector.
 131. The vector of claim 130, wherein the vector is alentiviral vector.
 132. An isolated T cell comprising the polynucleotideof claim 128 or the vector of any one of claims 129-131.
 133. Anisolated T cell comprising the CAR of any one of claims 112-127, whereinthe CAR is expressed on a cell surface of the T cell.
 134. The isolatedT cell of claim 132 or claim 133, wherein the T cell is a human T cell.135. A method for treating or preventing cancer in an individual,comprising administering to the individual an effective amount of acomposition comprising the T cell of any one of claims 132-134.
 136. Themethod of claim 135, wherein the cancer is selected from the groupconsisting of pancreatic cancer, colorectal cancer, esophageal cancer,leukemia, adenocarcinoma, fibrosarcoma, duodenal adenocarcinoma, braincancer, liver cancer, lung cancer, breast cancer, ovarian cancer, andcervical cancer.
 137. The method of claim 136, wherein the cancer isselected from the group consisting of pancreatic cancer, colorectalcancer, lung cancer, and brain cancer.
 138. The method of any one ofclaims 135-137, wherein the individual is a human.
 139. The method ofany one of claims 135-138, further comprising administering to theindividual an amount of another anti-cancer agent, whereby the T celland the anti-cancer agent in conjunction provide effective treatment orprevention of cancer in the individual.
 140. The method of claim 139,wherein the anti-cancer agent is a chemotherapeutic agent.